| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
b Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
calcium, fertilization, gamete biology, ovum, sperm
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Despite the breadth of these previous studies, information is lacking on the efficiency of fertilization of these treated eggs and on specific effects on gamete membrane interactions. Eggs from several mammalian species treated with cytochalasin B or D [1, 2, 4, 6, 15, 17] and from mouse treated with latrunculin A and jasplakinolide [3, 7] are capable of being fertilized. A few studies examine the efficiency of fertilization; cytochalasin B or latrunculin A treatments reduce the incidence of fertilization in sea urchin [1, 3] and porcine [8] eggs. We sought to extend these previous studies using ZP-free eggs to focus on gamete membrane interactions and using four different drugs (cytochalasin B, cytochalasin D, jasplakinolide, and latrunculin B) that perturb the actin cytoskeleton by three different mechanisms. In addition, cytochalasin D, jasplakinolide, and latrunculins have the advantage of having greater specificity than cytochalasin B, which inhibits glucose transporters in addition to perturbing actin microfilament polymerization [18].
In the present study, mouse eggs treated with cytochalasin D become more polyspermic than do control eggs, suggesting that cytochalasin D-treated eggs have an impaired ability to prevent the fusion of additional sperm. Blocks to polyspermy can occur at the level of the egg's extracellular matrix (the ZP), at the level of the egg plasma membrane, and perhaps also within the perivitelline space of ZP-intact eggs; different species rely on varying combinations of these blocks [19]. The ZP block to polyspermy is one of the primary egg activation responses to fertilization by the sperm, involving the exocytosis of cortical granules and the conversion of the ZP to a form that cannot support sperm binding [19, 20]. In contrast, little is known about the molecular basis of the membrane block in mammalian eggs, although evidence for its existence comes from several observations. These include a) observation of sperm in the perivitelline space of ZP-intact eggs that do not fuse with the egg plasma membrane (numerous studies, summarized in [19]), b) reduced penetration of sperm with ZP-free eggs that have been previously fertilized [21–25], c) a plateau in the number of sperm fusing with ZP-free eggs with increased time [21, 25], and d) a decrease in sperm-egg binding with increased time postinsemination or to previously fertilized eggs [26, 27]. However, it is not known if changes in the egg plasma membrane that lead to decreased ability to interact with sperm occur as a response of egg activation, occurring upon fertilization. Other egg activation responses, such as exit from metaphase II arrest and the ZP block to polyspermy (resulting from cortical granule exocytosis), are dependent on increases in cytosolic calcium and are blocked by the intracellular calcium chelator BAPTA-AM [28]. Our finding that BAPTA-AM-treated eggs become very polyspermic when inseminated suggests that the egg activation-induced increase in intracellular calcium concentration in the egg is necessary for the egg to establish the membrane block to polyspermy. Taken together, these data provide new insights into the possible modulation of receptivity of the mouse egg membrane to sperm after fertilization.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This work was conducted in accordance with the Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Throughout this manuscript, the term "egg" will be used to refer to female gametes at metaphase II. Eggs were obtained from superovulated CF-1 mice (Harlan, Indianapolis, IN) and cumulus cells were dispersed as previously described [29]. The ZP were removed by a very brief incubation (
15 sec) in acidic culture medium-compatible buffer (10 mM HEPES, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.4 mM KCl, 116.4 mM NaCl, final pH 1.5 [29]) and then allowed to recover for 60 min in Whitten medium [30] containing 15 mg/ml BSA (fatty acid-poor BSA from ICN, Costa Mesa, CA, or Albumax I from Gibco-BRL, Gaithersburg, MD). Eggs were cultured in 5% CO2 in air.
Following the ZP removal recovery period and prior to insemination, ZP-free eggs were incubated in cytochalasin B (Sigma, St. Louis, MO), cytochalasin D (Sigma), jasplakinolide (Molecular Probes, Eugene, OR), or latrunculin B (Calbiochem, La Jolla, CA) for 60 min. Two different lots of cytochalasin D were tested through the course of these experiments. Stock solutions of these drugs were made up in dimethylsulfoxide (DMSO) (cytochalasins B and D, 2 mg/ml; jasplakinolide, 1 mM; latrunculin B, 10 mM); DMSO was 99.9%. Final concentrations are indicated in the figure legends. For some experiments, eggs were treated with 10 µM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM; Calbiochem, La Jolla, CA; 10 mM stock in DMSO) following the ZP removal recovery period. ZP-free eggs were incubated in Whitten medium containing 15 mg/ml BSA and 10 µM BAPTA-AM for 60 min and then washed prior to insemination [28]. Culture medium for control eggs contained 1% DMSO. Control experiments confirmed that fertilization rates in medium containing and lacking 1% DMSO were identical (data not shown).
In vitro fertilization of ZP-free eggs was performed and assayed essentially as described previously. Insemination conditions (sperm concentrations and insemination times) were selected based on our previous studies [29]. Sperm were collected from the caudae epididymides of (C57BL/6J x SJL/J)F1 male mice (8–10 wk old; Jackson Laboratories, Bar Harbor, ME) or CD-1 male mice (retired breeders; Harlan) and cultured for 2.5–3 h in Whitten medium containing 15 mg/ml BSA to allow the sperm to capacitate and undergo spontaneous acrosome reactions. For experiments with actin-perturbing drugs, capacitated sperm were suspended in Whitten medium with 15 mg/ml BSA containing the indicated concentration of cytochalasin B, cytochalasin D, latrunculin B, jasplakinolide, or DMSO immediately prior to insemination. Actin perturbation induced by cytochalasin B, cytochalasin D, latrunculin B, and jasplakinolide is reversible, and thus eggs can recover from these treatments [7, 31]; therefore, the drugs were present during the insemination. Sperm were only exposed to an actin-perturbing drug for 5 min or less prior to addition to the start of insemination. Sperm were not exposed to any actin-perturbing drug during capacitation and thus were able to capacitate and undergo spontaneous acrosome reactions in the absence of any drug.
Eggs were inseminated in 10-µl culture medium drops, with 10 eggs per 10-µl insemination drop. The sperm concentration was 100 000 sperm/ml unless otherwise indicated. After the indicated insemination time, eggs were washed through three 100-µl drops of Whitten medium containing 15 mg/ml BSA with a thin-bore (
100 µm) pipette to remove loosely attached sperm. Sperm-egg binding and fusion was assessed as we have done previously [29, 32] by fixing the eggs in 3.7% paraformaldehyde in PBS and staining with 1.5 µg/ml 4',6'-diamidino-2-phenylindole to aid in the visualization and assessment of morphology of the sperm DNA. The presentation of values for average number of sperm bound per egg only includes sperm that were bound; sperm that had fused with the egg were excluded from these values. For some control experiments, egg actin microfilaments were stained with fluorescently labeled phalloidin (Sigma) as previously described [33].
Assessment of Acrosome Exocytosis in Sperm
Analyses were performed in two ways. For some experiments, cauda epididymal sperm were capacitated for 2.5–3.0 h in the presence of actin-perturbing drugs (control, 1% DMSO; or 5 µg/ml cytochalasin D, 20 µg/ml cytochalasin B, 0.35 µg/ml jasplakinolide, or 10 µg/ml latrunculin B). For other experiments, cauda epididymal sperm that had been capacitated for 2.5–3.0 h were then treated for 60 min with 5 µg/ml cytochalasin D, 20 µg/ml cytochalasin B, jasplakinolide, or latrunculin B (control, 1% DMSO) in medium lacking or containing 50 µM calcium ionophore A23187. Sperm were then fixed for 10 min with 70% ethanol, dried onto slides, and stained with 100 µg/ml peanut agglutinin-FITC (Vector Laboratories, Burlingame, CA) to stain the sperm acrosomes [34] and 0.5 µg/ml propidium iodide to stain sperm nuclei. Samples were viewed on a fluorescent microscope; 200 sperm were counted per sample to determine the percentage of acrosome-reacted sperm.
Sperm Motility Analysis by Computer-Assisted Semen Analysis
Sperm motility was assessed in a Hamilton Thorne Integrated Visual Optical System Sperm Analyzer (Hamilton Thorne Research, Beverly, MA) with parameters optimized for detection of mouse sperm. Sperm were diluted to 2–10 x 106/ml in Whitten medium containing 22 mM NaHCO3 and 15 mg/ml BSA and either 1% DMSO (control), 5 µg/ml cytochalasin D, or 20 µg/ml cytochalasin B. Analyses were performed at 30 and 90 min following the start of cytochalasin D or cytochalasin B treatment. Motility was tracked for 30 frames at a frame rate of 60 Hz. The percentage of motile sperm, the progressive velocity (the straight-line distance from the beginning to the end of each sperm's track divided by the time elapsed), the path velocity (the total distance along the smoothed average path for each sperm divided by the time elapsed), and the curvilinear velocity (the track speed, or total distance covered by each sperm divided by the time elapsed) were assessed. At least 100 sperm in each treatment group were analyzed per experiment.
Calcium Measurements
ZP-free eggs, treated as described with 20 µg/ml cytochalasin B or 5 µg/ml cytochalasin D, were loaded with Calcium Green-AM (Molecular Probes, Eugene, OR) by incubation in 10 µM Calcium Green-AM in Whitten medium containing 0.01% polyvinylalcohol (PVA) + 0.02% Pluronic F-127 (Molecular Probes) for 15–60 min. The eggs were then washed and cultured in Whitten medium containing 0.01% PVA until use.
For imaging of calcium oscillations, individual eggs were placed in a 90-µl drop of Whitten medium containing the appropriate additive (DMSO for controls or 20 µg/ml cytochalasin B or 5 µg/ml cytochalasin) under light mineral oil in a Leiden chamber (Medical Systems, Greenvale, NY) and allowed to settle on a glass coverslip coated with Cell-Tak (Collaborative Biomedical Products, Bedford, MA). The chamber was placed on a constant-temperature stage (Model 5000, Micro Devices, Newtown, PA) with a laminar flow of 5% CO2, 95% air over the chamber on an inverted microscope (TE 300, Nikon, Melville, NY). Once the egg had adhered to the coverslip, 10 µl of capacitated sperm (1–2 x 107/ml in Whitten medium containing 15 mg/ml BSA) were added. Final sperm concentration was 1–2 x 106/ml; it was necessary to use sperm concentrations in this range because some sperm would adhere to the Cell-Tak-coated coverslip. The egg was imaged using a 40x/0.75 numerical aperture objective, and the calcium green fluorescence was excited using a 100-watt mercury lamp and a fluorescein filter set. Emitted light was measured using a silicon photodiode (Model 71882, Oriel Instruments, Stratford, CT). The output from the photodiode was recorded on a chart recorder (Linear 1100, Barnstead/Thermolyne, Dubuque, IA).
Assay of ZP Conversion
ZP-intact eggs were collected and cultured in Whitten medium containing 5% fetal calf serum to prevent precocious ZP conversion [35]. Eggs were then cultured for 60 min in Whitten medium containing 5% fetal calf serum and either 1% DMSO (controls) or 20 µg/ml cytochalasin B or 5 µg/ml cytochalasin D. Eggs were washed through six drops of Whitten medium containing 15 mg/ml BSA prior to insemination to remove the fetal calf serum. Eggs were then inseminated for 2 h in Whitten medium containing 15 mg/ml BSA with 200 000 sperm/ml in the continued presence of 1% DMSO, 20 µg/ml cytochalasin B, or 5 µg/ml cytochalasin D. Control unfertilized eggs were cultured in parallel in Whitten medium containing 15 mg/ml BSA, with no fetal calf serum present. After 2 h, all eggs were washed and transferred to CZB medium [36] containing 5% fetal calf serum and then cultured overnight to allow pronuclei to form so that fertilized eggs (zygotes) could be selected for analysis of ZP conversion. (Because cytochalasins B and D inhibit cytokinesis, emission of the second polar body could not be used to assess whether an egg was fertilized or not.) Eggs that had been inseminated but lacked pronuclei were presumed to be unfertilized and were not used for ZP analysis. Assessment of ZP2 conversion to ZP2f was performed as previously described [37].
Statistical Analysis
Errors bars in all figures represent the SEM. Statistical analysis was performed using Statview 5.0 (SAS Institute, Cary, NC). The pooled data from each series of experiments were analyzed for differences between all of the treatment groups by ANOVA and, when indicated by ANOVA results, by Fisher protected least significant difference post hoc testing to evaluate pairwise differences (P < 0.05 considered significant).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To examine the effects of actin-disrupting drugs on gamete membrane interactions and fertilization, ZP-free eggs were treated prior to insemination and then inseminated with 100 000 sperm/ml for 90 min in the presence of the following drugs: cytochalasin D (2.5, 5.0, and 10.0 µg/ml, corresponding to 4.93, 9.85, and 19.7 µM, respectively), cytochalasin B (5, 10, and 20 µg/ml, equivalent to 10.45, 20.9, and 41.8 µM, respectively), jasplakinolide (0.07, 0.175, 0.35, and 0.7 µg/ml, corresponding to 100, 250, 500, and 1000 nM, respectively), or latrunculin B (0.8, 4, 10, and 20 µg/ml, corresponding to 2, 10, 25, and 50 µM, respectively). The four drugs were selected because they perturb the actin cytoskeleton by three different mechanisms. Cytochalasins B and D bind to the fast-growing (barbed) ends of microfilaments, blocking the addition of monomeric actin to microfilaments and reducing actin polymerization [18]. Cytochalasin D is more effective than cytochalasin B, and cytochalasin B has an additional effect, inhibiting glucose transport [18]. Latrunculins A and B bind to monomeric actin, preventing its addition to microfilaments and thus blocking actin polymerization [31, 38]; there is also evidence that latrunculin A competes with thymosin ß4 for binding actin monomers in Xenopus eggs [12]. Jasplakinolide, in contrast, stabilizes existing actin microfilaments apparently by a mechanism similar to phalloidin, through binding to microfilaments and reducing depolymerization as well as increasing polymerization [18, 39, 40]. We confirmed that these treatments affected the mouse egg actin cytoskeleton as previously reported by staining eggs with fluorescently labeled phalloidin. Cytochalasin D, cytochalasin B, and latrunculin B at all concentrations caused a dramatic decrease in phalloidin staining, including a reduction or disappearance of the actin-rich cap over the spindle in metaphase II eggs, and failure to emit the second polar body or form fertilization cones over sperm heads in fertilized eggs (data not shown), in agreement with prior work [1–3]. Jasplakinolide-treated eggs had bundles and aggregates of phalloidin-stained filaments and did not emit polar bodies or form fertilization cones, as observed previously [7].
Treatment of eggs with cytochalasin B, jasplakinolide, and latrunculin B resulted in a decrease in the incidence of fertilization (Fig. 1). Eggs were fertilized in all treatment groups, but the average number of sperm fused per egg, the percentage of fertilized eggs, and the percentage of polyspermic eggs were decreased in eggs treated with these three pharmacologic agents. The lowest levels of fertilization were observed in the eggs treated with the highest concentrations of these drugs. In contrast, zygotes resulting from cytochalasin D-treated eggs showed an increase in polyspermy compared with DMSO-treated controls. This effect on the extent of polyspermy was most obvious in eggs treated with 2.5 and 5 µg/ml cytochalasin D. The average number of sperm fused per egg was increased in eggs treated with 5 µg/ml of cytochalasin D compared with controls (Fig. 1A), and the percentage of polyspermic eggs in the 5 µg/ml cytochalasin D treatment group was double that of matched controls (48% versus 25%; Fig. 1B). In addition, the fertilized cytochalasin D-treated eggs tended to be more polyspermic, having more decondensing sperm heads within the egg cytoplasm (1.74 or 1.80 sperm per fertilized egg treated with 2.5 or 5 µg/ml cytochalasin D, respectively; these differences were statistically significant compared with the 1.38 sperm per fertilized control egg; P < 0.01; Fig. 1B). All zygotes resulting from control eggs and eggs treated with the actin-disrupting drugs had resumed meiosis by this 1.5-h postinsemination time point. In these zygotes, the metaphase-to-anaphase transition was viewed by staining of the maternal DNA with DAPI (data not shown). No qualitative differences in sperm motility were observed in any of the treatment groups (cytochalasin D, cytochalasin B, jasplakinolide, or latrunculin) at any of the time points during the insemination.
|
The occurrence of spontaneous and A23187-induced acrosome reactions in capacitated sperm was not affected by actin-disrupting drugs (Fig. 2). Exposure of cauda epididymal sperm to actin-disrupting drugs during the 2.5–3-h capacitation time also did not alter the rates of spontaneous acrosome reactions (control, 50 ± 13% acrosome-reacted; 5 µg/ml cytochalasin D, 57 ± 13%; 20 µg/ml cytochalasin B, 49 ± 15%; 0.35 µg/ml jasplakinolide, 43 ± 16%; 10 µg/ml latrunculin B, 41 ± 17%; no statistically significant differences; n = 3 experiments).
|
To investigate further the means by which cytochalasin D led to an increase in polyspermy, we performed additional experiments comparing the effects of cytochalasin D and cytochalasin B on gamete membrane interactions. Cytochalasin B was selected for comparison with cytochalasin D because it is a structurally similar fungal toxin that perturbs the actin cytoskeleton by similar mechanisms and yet has very different effects on murine fertilization (Fig. 1). We used cytochalasin D at 5 µg/ml (CD-5) and cytochalasin B at 20 µg/ml (CB-20) because these had the most dramatic effects of the concentrations examined (Fig. 1).
To determine if differences in the timing of sperm-egg fusion after treatment of eggs with CD-5 or CB-20 could explain the results in Figure 1, treated eggs were examined at a series of time points after insemination (Fig. 3). The rationale for these experiments was to assess if sperm-egg fusion was decreased or delayed in the CB-20 treatment group and if sperm-egg fusion was increased or accelerated in the CD-5 treatment group compared with controls. As shown in Figure 3, the average number of sperm fused per egg for control eggs plateaued with increased time. In CB-20-treated eggs, the average number of sperm fused per egg was lower than for control eggs and was not statistically different in CB-20-treated eggs at the 1.5- and 4-h postinsemination time points (Fig. 3). This suggested that the CB-20 treatment did not simply delay gamete fusion because increased time did not lead to increased incidence of fertilization. Fewer of the fertilized CB-20-treated eggs (29.6%) had maternal and/or paternal DNA in the early stages of pronuclear development compared with control eggs (62.2%) at the 4-h time point. Very few of the fertilized CB-20-treated eggs were polyspermic (2/54 eggs from three separate experiments). In fertilized CD-5-treated eggs, the average number of sperm fused per egg was initially similar to controls (0.75 h postinsemination) and then increased at the 1.5-, 2.5-, and 4.0-h time points (Fig. 3); the differences at these time points between CD-5 and controls are statistically significant (P < 0.05). Pronuclear formation appeared to be similar in zygotes resulting from control and CD-5-treated eggs; 62.2% and 68.8% of the fertilized control and CD-5-treated eggs, respectively, had progressed to the early stages of pronuclear development by 4-h postinsemination. Additionally, in polyspermic zygotes in the control and CD-5 groups, sperm could be observed in varying stages of nuclear decondensation and/or pronuclear formation within the zygote cytoplasm at this 4-h time point. In other words, one sperm within the cytoplasm of a polyspermic zygote would be in the early stages of pronuclear formation while another sperm would be in the earlier stages of nuclear decondensation (data not shown). This observation implied that sperm-egg fusion could have occurred asynchronously, with some sperm fusing with the egg at later time points than others.
|
No qualitative differences in sperm motility were observed in any of the treatment groups (cytochalasin D, cytochalasin B, jasplakinolide, or latrunculin). In addition, we used computer-assisted semen analysis to assess the effects of CD-5 and CB-20 treatments on sperm motility more precisely. The rationale for this was that the decrease in sperm-egg fusion in the CB-20 treatment group (Figs. 1 and 3) or the increase in sperm-egg fusion in the CD-5 treatment group (Figs. 1 and 3) might be due to changes in sperm motility. Capacitated sperm were diluted in Whitten medium containing either 1% DMSO (control), 5 µg/ml cytochalasin D, or 20 µg/ml cytochalasin B, and motility analyses were performed at 30 and 90 min following the start of cytochalasin D or cytochalasin B treatment. The percentages of motile sperm were not statistically different in the three treatment groups at 30 or 90 min (data not shown). The progressive velocity (the straight-line distance from the beginning to the end of each sperm's track divided by the time elapsed), the path velocity (the total distance along the smoothed average path for each sperm divided by the time elapsed), and the curvilinear velocity (the track speed, or total distance covered by each sperm divided by the time elapsed) also were not statistically different in the three treatment groups at 30 and 90 min, with one exception. Progressive motility in the CB-20-treated sperm was decreased 25% compared with control sperm after 90 min of treatment (P < 0.05) (Fig. 4).
|
To determine if the differences in sperm-egg fusion were due to alterations in sperm-egg plasma membrane binding, we examined the numbers of sperm bound (not fused) per egg, fused per egg, and total egg-associated (bound or fused) sperm per egg after insemination for 90 min with 100 000 sperm/ml (Table 1). The number of sperm fused per CD-5-treated eggs was higher than controls, and the number of sperm fused per CB-20-treated egg was lower than controls, similar to data in Figure 1. The numbers of total egg-associated and bound sperm to CD-5-treated and CB-20-treated eggs was reduced compared with controls. The initial contacts and attachments of sperm to eggs (observed with a dissecting microscope during the insemination) appeared to be similar in all three groups, but after 90 min insemination, the number of sperm that attached firmly enough to remain bound to the egg and not be removed by washes was lower in CD-5- and CB-20-treated eggs compared with controls. In addition, control eggs had a ratio of 0.16 fused sperm per total number of all egg-associated sperm (i.e., sperm that were bound to or fused with the egg), whereas the CD-5-treated eggs had a ratio of 0.96 fused sperm per total egg-associated sperm (Table 1). This indicates that virtually all of the sperm associated with the CD-5-treated eggs fuse with the eggs, whereas approximately one out of six sperm associated with the control eggs proceed to sperm-egg fusion.
|
Two series of experiments were performed in which eggs that were pretreated and then inseminated in the presence of CD-5 or CB-20 were compared with eggs that were only inseminated in the presence of CD-5 or CB-20, without the 60-min pretreatment. In one experimental series, eggs were inseminated for 15 min with 500 000 sperm/ml, similar to previous assays of sperm-egg binding [26, 27] in which the rationale for examining earlier postinsemination time points was that this time is prior to the establishment of the membrane block to polyspermy. (Please note that, at 15 min postinsemination, the number of bound sperm and total egg-associated sperm was the same; sperm-egg fusion was not detected at this early time point.) In these experiments, there was a dramatic decrease in sperm-egg binding in eggs treated with CD-5 for 60 min and then during the 15-min insemination (a total of 75 min) compared with controls. Eggs that were treated with CD-5 only for the 15-min insemination showed sperm-egg binding levels that were lower than controls but higher than eggs that were treated with CD-5 for 60 min prior to insemination and during the 15-min insemination (Table 2). The CB-20-treated eggs (either pretreated and treated during insemination or during insemination only) showed a number of bound sperm that was not different from the number of bound sperm to control eggs (Table 2). The differences here between cytochalasins B and D could be due to the greater potency of cytochalasin D [18]. In a second experimental series, eggs were inseminated for 45 min with 125 000 sperm/ml. There were no statistically significant differences in sperm-egg fusion values, although total egg-associated sperm and bound sperm values were affected. CD-5- and CB-20-treated eggs, either pretreated and treated during insemination (total 105-min exposure to drug) or treated only during insemination (45-min exposure), showed a decrease in the number of bound sperm and total-egg associated sperm compared with control eggs (Table 2). There were no statistically significant differences in these values (total sperm or bound sperm) between the eggs that were pretreated and then inseminated in the presence of drug and the eggs that were only exposed to drug during the insemination. The differences between comparable CD-5-treated and CB-20-treated samples (pretreatment + insemination exposure, or insemination only exposure to drugs) were not significant.
|
The effect of sperm concentration used in the insemination on the extent of polyspermy was also examined by inseminating control and CD-5-treated eggs with 50 000, 100 000, or 300 000 sperm/ml for 1.5 h (Fig. 5). There was a significant increase (P < 0.05) in the average number of sperm fused per CD-5-treated egg compared with controls for each of the three different sperm concentrations tested, further demonstrating that the extent of polyspermy was increased in CD-5-treated eggs compared with controls. The average numbers of sperm fused per egg in CD-5-treated eggs inseminated with the three sperm concentrations were not statistically different. In control eggs, the average number of sperm per egg for eggs inseminated with 300 000 sperm/ml was not statistically different from the values for eggs inseminated with 50 000 or 100 000 sperm/ml (the value for eggs inseminated 100 000 sperm/ml was different from the value for eggs inseminated with 50 000 sperm/ml but not 300 000 sperm/ml). There did not appear to be a significant trend toward increased polyspermy with increased inseminating sperm concentrations in either control or CD-5-treated eggs.
|
The data in Figures 1, 3, and 5 and Table 1 indicate that CD-5-treated eggs become more polyspermic than do control eggs, suggesting that CD-5 treatment could impair the ability of the egg to establish a membrane block to polyspermy. The molecular basis of the membrane block in mammalian eggs is not known, although evidence for its existence comes from a variety of observations (see Introduction). We hypothesized that the membrane block to polyspermy was likely to be established as a component of the egg activation response to fertilization and that CD-5 treatment led to an increase in polyspermy by impairing egg activation. Therefore, we examined whether CD-5 treatment impaired two specific egg activation responses: the initiation and/or perpetuation of oscillations in cytosolic calcium concentrations ([Ca2+]cyt) and ZP2 conversion to ZP2f following fertilization, which is the basis of the ZP block to polyspermy and has been correlated with the extent of cortical granule exocytosis [41]. We examined CB-20 treatment in these studies as well.
There were no differences between the sperm-induced calcium oscillations induced by sperm in control eggs, CD-5-treated eggs, and CB-20-treated eggs (Fig. 6). Specifically, the mean time to the initiation of the first calcium oscillation, the duration of the first oscillation, and the times between oscillations were not statistically different in comparisons among treatment groups. Based on these observations, calcium transients appear to be temporally normal in CD-5-treated eggs and CB-20-treated eggs. We also examined the extent of ZP2 conversion to ZP2f in fertilized CD-5- and CB-20-treated eggs. There were two reasons for this. First, ZP conversion is a result of cortical granule exocytosis, an egg activation response that is dependent on an increase in [Ca2+]cyt in the egg [28]. Second, cortical granule exocytosis and conversion of the ZP to a form that cannot be bound by sperm has been proposed to be dependent on the actin cytoskeleton, based on studies using cytochalasin B on hamster eggs [10]. To assess if CD-5 or CB-20 treatment of mouse eggs affected ZP conversion, ZP-intact eggs were incubated and then inseminated in the presence of CD-5 or CB-20. We found that amounts of ZP2f were not statistically different in control and CD-5 treatment groups (Fig. 6C). CB-20 had a moderate inhibitory effect on ZP conversion, resulting in a decrease in the percentage of ZP2 proteins that had been converted to ZP2f. The difference in the amount of ZP2f was statistically significant in the CB-20 group compared with the fertilized control eggs (treated with the solvent, DMSO) and with unfertilized eggs (P < 0.05).
|
The observation that CD-5-treated eggs show normal Ca2+ oscillations upon fertilization (Fig. 6A) suggested that increases in [Ca2+]cyt were not sufficient for the membrane block to polyspermy to be established. In order to determine if an increase in [Ca2+]cyt was required for the membrane block to polyspermy to be established, we treated eggs with the intracellular Ca2+ chelator, BAPTA-AM, which at 10 µM suppresses all Ca2+ oscillations that normally occur upon fertilization [28]. We found that BAPTA-AM-treated eggs become significantly more polyspermic than do control eggs. In experiments in which we assessed sperm-egg fusion at various times (0.75, 1.5, and 4.0 h) after insemination with 100 000 sperm/ml (Fig. 7A), control eggs gradually plateaued in their average sperm-fused-per-egg values with increased time (seen also in the experiments in Fig. 2). BAPTA-AM-treated eggs showed dramatically higher levels of sperm-egg fusion that increased with time (Fig. 7A) as well as increased levels of polyspermy compared with control eggs when inseminated for 1.5 h with three different sperm concentrations (Fig. 7B). In addition, there was a correlation between increased polyspermy and increased inseminating sperm concentrations in the BAPTA-AM-treated eggs, as the differences in the average sperm fused per BAPTA-AM-treated egg values between the three sperm concentrations were statistically significant (P < 0.05). In control eggs, the average number of sperm fused per egg in eggs inseminated with 300 000 sperm/ml was statistically higher than the values for control eggs inseminated with 50 000 or 100 000 sperm/ml, although the value for eggs inseminated with 100 000 sperm/ml was not statistically different from the value for eggs inseminated with 50 000 sperm/ml. There did not appear to be a noteworthy trend toward increased polyspermy with increased inseminating sperm concentrations in control eggs, similar to what was observed with Figure 3. It should be noted that treatment of eggs with BAPTA-AM did not cause overt disruption or reorganization of the actin cytoskeleton; phalloidin staining of control and BAPTA-AM-treated eggs was similar, revealing actin-rich caps over the meiotic spindles in unfertilized eggs and fertilization cones over decondensing sperm heads in fertilized eggs (data not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
30–60 min postinsemination [26, 27]. The ability of fertilized eggs to be penetrated by sperm is reduced by 45–90 min postinsemination, and the ability of two-cell embryos to fuse with sperm is very low [22, 26, 48]. The low penetrability of two-cell embryos could be due to a persisting membrane block and/or incorporation of a new "infusible" membrane during embryonic cleavage; in a similar fashion, the introduction of infusible membrane (from embryos) to an egg by cell fusion results in a hybrid cell that fuses poorly with sperm [49]. Parthenogenetically activated eggs or eggs that have been fertilized by intracytoplasmic sperm injection are capable of undergoing sperm-egg fusion when inseminated, and thus appear to have a reduced or absent membrane block to polyspermy [23, 50–53]. These observations suggest that the membrane block to polyspermy appears to be induced by or to require normal fertilization by a sperm. Cortical granule exocytosis does not appear to be sufficient to establish the membrane block because eggs activated by artificial stimuli or by intracytoplasmic sperm injection undergo cortical granule exocytosis and yet become polyspermic when inseminated [23, 50–53]. Our data suggest that the actin cytoskeleton participates in the membrane block to polyspermy in mouse eggs and that this process also requires increases in [Ca2+]cyt that occur upon egg activation with fertilization. Considering the key roles that calcium plays in egg activation and that the actin cytoskeleton plays in cell morphology, interactions with membranes and membrane proteins, organelle transport, signaling, and other processes, these findings are not surprising. The actin microfilaments, in conjunction with other associated proteins, interact with the cytoplasmic domains of integrins in other cell types [54]. With increasing insight into the involvement of egg membrane proteins such as members of the integrin family [55–58] and the integrin-associated protein CD9 [32, 58–63] in gamete membrane interactions, the cortical actin cytoskeleton of the egg is an excellent candidate to play a role in modulating gamete membrane interactions. Ultrastructural analyses of cortical preparations from hamster eggs undergoing fertilization show that microfilaments in the hamster egg cortex are associated with the fusing sperm [64]. The cortical cytoskeleton of the egg could also affect changes in egg membrane fluidity or the incorporation or diffusion of the sperm membrane components into the egg plasma membrane, two postfertilization events that might be correlated with the establishment of a membrane block to polyspermy [47, 52, 53]. The identification of the calcium dependence of the membrane block to polyspermy places it with other egg activation responses, such as cortical granule exocytosis and cell cycle resumption and exit from metaphase II arrest. Both of these processes are disrupted in BAPTA-AM-treated eggs [28], as is the establishment of the membrane block to polyspermy (Fig. 7). We can also conclude that calcium signaling is necessary but not sufficient since CD-5-treated eggs have normal temporal patterns of calcium oscillations upon fertilization and yet become more polyspermic than do controls, which have similar calcium oscillations (Fig. 6A).
While treatment of eggs with cytochalasin D leads to an increase in the incidence of sperm-egg fusion, this treatment did not enhance other gamete functions that precede sperm-egg fusion. Cytochalasin D did not affect sperm motility (Fig. 4) or spontaneous or calcium ionophore-induced acrosome reactions in capacitated sperm (Fig. 2). Sperm-egg binding was also not increased; in fact, sperm-egg binding to CD-5-treated eggs was lower than to control eggs (Tables 1 and 2), resulting in an increase in the ratio of the sperm that fused with the egg to the total number of egg-associated sperm (Table 1). Furthermore, experiments examining the time course of fertilization indicate that levels of sperm-egg fusion in CD-5-treated eggs initially match those of control eggs (up to 0.75 h postinsemination) and then are higher at later time points (Fig. 3). In contrast, levels of sperm-egg fusion in control eggs increase from 0–1.5 h but then plateau (Figs. 3 and 7A); control eggs do not catch up to CD-5-treated eggs. These data are also in agreement with studies suggesting that the membrane block to polyspermy in mouse eggs is established around 40 min to 2 h postinsemination [21, 23, 27].
The increase in the extent of polyspermy occurs in eggs treated with cytochalasin D but not with the other three drugs tested in this study. Two drugs, latrunculin B and jasplakinolide, that perturb the actin cytoskeleton by mechanisms different from that of cytochalasin D do not have this effect of increased polyspermy in treated eggs and in fact decrease the incidence in sperm-egg fusion (Fig. 1). This may be because the disruption of the actin cytoskeleton by these drugs renders the egg membrane environment unable to support sperm interactions. Cytochalasin B also does not increase the extent of polyspermy (Figs. 1 and 3) even though cytochalasin B impedes actin polymerization by binding to the barbed ends of actin microfilaments, as cytochalasin D does. Similar to cytochalasin D, cytochalasin B treatment of eggs leads to a decrease in sperm-egg binding (Tables 1 and 2); these effects may be due to perturbation of the egg actin cytoskeleton affecting the ability of the membrane to support sperm binding. However, unlike cytochalasin D, cytochalasin B causes a slight decrease in progressive sperm motility (Fig. 4) and a decrease in sperm-egg fusion (Fig. 1). Experiments examining the time course of fertilization indicate that the levels of sperm-egg fusion to CB-20-treated eggs does not increase with longer insemination times, suggesting that the decrease in sperm-egg fusion observed at 90 min postinsemination is not due to delays in sperm-egg fusion (Fig. 3). It is not clear why cytochalasin D and cytochalasin B have such different effects, although possibilities are that cytochalasin B is known to be less potent than cytochalasin D in its perturbation of the actin cytoskeleton and/or that cytochalasin B also inhibits glucose transporters [18]. Mammalian eggs and sperm express glucose transporters, and glucose transport in these cells is inhibited by cytochalasin B [65–68], raising the possibility that the reduction of sperm motility and of ZP conversion through cortical granule exocytosis by cytochalasin B was due, at least in part, to inhibition of glucose transporters.
The picture of how a mouse egg establishes a membrane block to polyspermy is still incomplete, but the data presented offer some important advances. The results with BAPTA-AM demonstrate the importance of calcium signaling for the establishment of the membrane block (Fig. 7). The increase in [Ca2+]cyt upon fertilization is necessary but is not sufficient, however. CD-5-treated eggs become polyspermic and yet have normal temporal patterns of calcium oscillations (Fig. 6A), although it is possible that subtle alterations in the calcium oscillations could exist in CD-5-treated eggs and may affect membrane dynamics. Calcium signaling may affect multiple components of the establishment of the membrane block to polyspermy, one component of which could be the actin cytoskeleton, based on the observation that BAPTA-AM-treated eggs tend to be more polyspermic than do CD-5 treated eggs (Figs. 3 and 7). The actin cytoskeleton could participate in the membrane block by modulating membrane receptivity, leading to a decrease in sperm-egg adhesion and/or decreasing the capacity of the egg membrane to fuse with the sperm membrane. Cytochalasin D may perturb the latter process, as CD-5-treated eggs show increased ratios of fused sperm to total egg-associated sperm (Table 1). Cortical granule exocytosis does not appear to be sufficient for the membrane block ([23, 50, 51]; Fig. 6). Because eggs activated by parthenogenetic stimuli [23, 50, 51] or fertilized by intracytoplasmic sperm injection [52, 53] have a reduced ability to establish the membrane block to polyspermy, normal gamete membrane interactions and/or events associated with them may be required for the membrane block. Possibilities include incorporation of the sperm membrane, changes in the actin cytoskeleton and/or other cortical structures associated with sperm incorporation, and proper spatial organization of sperm-induced signal transduction. With these insights, progress can be made toward characterizing both upstream and downstream events related to postfertilization membrane and cortical dynamics in mammalian eggs.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Janice P. Evans, Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Room 3606, 615 N. Wolfe Street, Johns Hopkins University, Baltimore, MD 21205. FAX: 410 614 2356; jpevans{at}jhsph.edu
Received: 15 February 2002.
First decision: 5 March 2002.
Accepted: 3 May 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(ADAM1)-mediated sperm-egg cell adhesion during fertilization and identification of an adhesion-mediating sequence in the disintegrin-like domain. J Biol Chem 2001 276:24937-24945
