This Article Abstract Full Text (PDF) Supplementary Figure Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gardner, A. J Articles by Evans, J. P Search for Related Content PubMed PubMed Citation Articles by Gardner, A. J Articles by Evans, J. P

Establishment of the mammalian membrane block to polyspermy: evidence for calcium-dependent and -independent regulation

¹ Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Room W3606, 615 N. Wolfe St., Maryland, USA and ² Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women’s Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

Correspondence should be addressed to J P Evans; Email: jpevans{at}jhsph.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
One crucial result of egg activation is the establishment of blocks on the zona pellucida and the egg plasma membrane to prevent fertilization by additional sperm. The mechanism(s) by which a mammalian egg regulates the establishment of the membrane block to polyspermy is largely unknown. Since Ca²⁺ signaling regulates several egg activation events, this study investigates how sperm-induced Ca²⁺ transients affect the membrane block to polyspermy, building on our previous work (Biology of Reproduction 67:1342). We demonstrate that mouse eggs that experience only one sperm-induced Ca²⁺ transient establish a membrane block that is less effective, than in eggs that experience normal sperm-induced Ca²⁺ transients but that is more effective than in eggs with completely suppressed [Ca²⁺]_cyt increases. Sperm-induced increases in [Ca²⁺]_cyt regulate the timing of membrane block establishment, as this block is established more slowly in eggs that experience one or no sperm-induced Ca²⁺ transients. Finally, our studies produce the intriguing discovery that there is also a Ca²⁺-independent event that is associated with fertilization in the pathway leading to membrane block establishment. Taken together, these data indicate that Ca²⁺ plays a role in facilitating membrane block establishment by regulating the timing with which this change in egg membrane function occurs, and also that the membrane block differs from other post-fertilization egg activation responses as Ca²⁺ is not the only stimulus. The membrane block to polyspermy in mammalian eggs is likely to be the culmination of multiple post-fertilization events that together modify the egg membrane’s receptivity to sperm.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Calcium signaling plays a key role in the earliest stages of mammalian development, with transient increases (also called oscillations) in cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) in fertilized eggs inducing a cascade of cellular changes collectively known as egg activation (Runft et al. 2002, Malcuit et al. 2006). The egg activation responses triggered by Ca²⁺ signals serve to initiate embryonic development and to prevent polyspermy (fertilization by additional sperm). The repetitive transient increases in [Ca²⁺]_cyt allow for the different events of mammalian egg activation to be encoded in a specific temporal order by the same upstream stimulus, as the characteristics of Ca²⁺ transients appear to be ‘decoded’ to generate multiple cellular responses (Ducibella et al. 2002, Berridge et al. 2003).

The goal of this study is to investigate how sperm-induced Ca²⁺ transients associated with egg activation affect the membrane block to polyspermy. Mammalian eggs use both a zona pellucida (ZP) block and a membrane block to prevent polyspermy (Yanagimachi 1994, Abbott & Ducibella 2001). Very little is known about the membrane block, although evidence demonstrating its existence comes from several studies (Austin 1961, Wolf 1978, Zuccotti et al. 1991, Horvath et al. 1993, Maluchnik & Borsuk 1994, Sengoku et al. 1995, McAvey et al. 2002, Gardner & Evans 2006). The mechanism by which a mammalian egg membrane decreases its receptivity to sperm is not known. While non-mammalian species (i.e. frogs, several marine invertebrates) use a membrane block that involves a rapid and transient depolarization of egg membrane potential (Jaffe & Gould 1985, Gould & Stephano 2003), it appears that the mammalian membrane block works by a different mechanism as membrane depolarization is not observed in fertilized mouse, hamster, or rabbit eggs (Miyazaki & Igusa 1981, Igusa et al. 1983, Jaffe et al. 1983, McCulloh et al. 1983).

The mechanism by which a mammalian egg regulates the establishment of the membrane block also is largely unknown. Our previous work indicates that increased [Ca²⁺]_cyt is an important component of membrane block establishment, as complete suppression of sperm-induced Ca²⁺ transients with 10 µM BAPTA-AM, an intracellular Ca²⁺ chelator, results in a dramatic increase in polyspermy in ZP-free mouse eggs (McAvey et al. 2002). Other events of egg activation such as cortical granule (CG) exocytosis and cell cycle resumption, also do not occur in eggs in which increased [Ca²⁺]_cyt is completely inhibited. It is further known from a variety of studies, such as those using either partial suppression of sperm-induced Ca²⁺ transients or parthenogenetic activation resulting from different types of manipulated [Ca²⁺]_cyt increases, that different egg activation responses require different ‘levels’ of calcium-mediated signals for initiation and then completion (Kline & Kline 1992, Ducibella et al. 2002, Ozil et al. 2005). For example, studies using electropermeabilization of mouse eggs to induce pulses of Ca²⁺ influx that mimic post-fertilization Ca²⁺ transients show that one pulse is sufficient to initiate CG exocytosis, 4–8 pulses in mouse eggs induce exit from metaphase II arrest and pronuclear formation requires 8–24 pulses (Ducibella et al. 2002). Thus, we sought to define more clearly the Ca²⁺ dependence of membrane block establishment. Work presented here shows that mouse eggs that experience only one sperm-induced Ca²⁺ transient establish a membrane block that is intermediate in effectiveness – i.e. less effective at preventing multiple fertilizations than in eggs that experience normal sperm-induced Ca²⁺ transients, but more effective than in eggs treated with 10 µM BAPTA-AM to suppress Ca²⁺ transients completely.

Another significant unknown is precisely how the membrane blocks in eggs experiencing little or no sperm-induced [Ca²⁺]_cyt increase are deficient in preventing additional fertilization. Insights into this mechanism would shed light on what role Ca²⁺ plays in membrane block establishment. We test the following possibilities: (1) the membrane block is less effective at preventing additional sperm fusion; (2) the membrane block is established more slowly and thus additional sperm have a larger time window to fertilize the egg after the first sperm has fused; or (3) the membrane block is transient and thus additional sperm can penetrate the egg after the initial block weakens. The studies here demonstrate that sperm-induced increases in [Ca²⁺]_cyt regulate the timing of membrane block establishment. This work also produced the interesting finding; there is a Ca²⁺-independent component in the pathway that contributes to membrane block establishment. These data suggest that Ca²⁺ plays a role in facilitating membrane block establishment, and that the membrane block is fundamentally different from other post-fertilization egg activation responses as Ca²⁺ is not the only driver.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Egg collection, BAPTA-AM treatment, and in vitro fertilization
Egg collection was performed as previously described (McAvey et al. 2002). Metaphase II-arrested eggs were collected from 6 to 8-week-old-superovulated CF-1 mice (Harlan, Indianapolis, USA) at 13 h after human chorionic gonadotropin (hCG) injection unless otherwise indicated. Cumulus cells were removed by brief incubation (< 5 min) in either Whitten’s medium (109.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5.5 mM glucose, 0.23 mM pyruvic acid, 4.8 mM lactic acid hemicalcium salt; Ca²⁺ concentration is 2.4 mM (Whitten 1971)) with 7 mM NaHCO₃ and 15 mM HEPES (hereafter referred to as ‘Whitten’s-HEPES’) and 0.04% Type I-S hyaluronidase (Sigma), or in Whitten’s-HEPES medium containing 30 mg/ml bovine serum albumin (BSA; Albumax I from Gibco-BRL) and 0.02% Type IV-S hyaluronidase (Sigma). After cumulus cell removal, the ZP were removed by a brief incubation (~15 s) in acidic culture medium compatible buffer (10 mM HEPES, 1 mM NaH₂PO₄, 0.8 mM MgSO₄, 5.4 mM KCl, 116.4 mM NaCl; pH 1.5) and then the eggs were allowed to recover for 60 min in Whitten’s medium containing 22 mM NaHCO₃ and 15 mg/ml BSA. Eggs were cultured in a humidified atmosphere of 5% CO₂ in air. Following the recovery period and prior to insemination, ZP-free eggs were incubated in Whitten’s medium containing 15 mg/ml BSA and either 0.5, 1, 2, or 5 µM 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM; Calbiochem, La Jolla, CA, USA; 10 mM stock in DMSO) for 60 min, then washed before insemination. Culture medium for control eggs contained 0.1% DMSO.

In vitro fertilization (IVF) of ZP-free eggs was performed essentially as described previously (Evans et al. 1995, McAvey et al. 2002) with the following additions. Sperm from one epididymis of a killed CD1 male mouse (8-week-old or retired breeders, Harlan) was collected in 125 µl Whitten’s medium containing 15 mg/ml BSA. After 10–15 min, tissue was removed from the droplet and the sperm were pipetted into the bottom of a tube containing 750 µl Whitten’s medium containing 15 mg/ml BSA (‘swim-up’). After 45 min, 225 µl from the top of the swim-up culture were removed and placed in a culture dish and covered with light mineral oil. The sperm were cultured for a total of 2.5–3 h in Whitten’s medium containing 15 mg/ml BSA to allow the sperm to undergo capacitation and spontaneous acrosome exocytosis. Ten eggs per 10 µl drop were inseminated for 0.75, 1.5 or 4 h with a sperm:egg ratio of 50:1 (50 000 sperm/ml) unless otherwise noted. These insemination conditions for the assessment of the membrane block to polyspermy (i.e. sperm incorporation over time) were chosen to achieve 100% fertilization in control eggs; this resulted in ~1–2.5 sperm fused per egg. After the indicated insemination time, the eggs were washed through three drops of Whitten’s medium containing 15 mg/ml BSA using a thin bore pipette to detach any loosely attached sperm. Eggs were then fixed in 3.7% paraformaldehyde in PBS and stained with 1.5 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) to assess the stage of maternal DNA and visualize decondensing sperm heads in the egg cytoplasm.

Calcium imaging
For analysis of sperm-induced [Ca²⁺]_cyt in eggs treated with BAPTA-AM, eggs were treated with 0, 0.5, 1, 2, 5, or 10 µM BAPTA-AM as described above. Just prior to imaging, ZP-free eggs were loaded with fura-2, acetoxymethyl ester (10 µM in Whitten’s medium containing 15 mg/ml BSA and 0.025% Pluronic F-127; Molecular Probes, Eugene, OR, USA) at 37 °C in an atmosphere of 5% CO₂ in humidified air for 20 min. Inseminations were performed by placing 8–10 eggs from each treatment group in a 5 µl drop of Whitten’s medium (without BSA) under light mineral oil in a Leiden chamber (Medical Systems, Greenvale, NY, USA), allowing the eggs to settle on a glass coverslip. The chamber was placed on a constant temperature stage (Model 5000, Micro Devices, Newtown, PA, USA) on an inverted microscope (TE 300, Nikon, Melville, NY, USA), with a laminar flow of 5% CO₂ in air over the chamber. Once the eggs adhered to the coverslip, 5 µl Whitten’s medium containing 30 mg/ml BSA were added to bring the final concentration of BSA to 15 mg/ml. Two microliters of capacitated epididymal sperm of 6-week-old B6SJLF₁/J males (Jackson Laboratories, Bar Harbor, ME; 1–2 x 10⁶ sperm/ml in Whitten’s medium containing 15 mg/ml BSA) were then added to the adherent eggs in the 10 µl medium.

For analysis of [Ca²⁺]_cyt in Ca²⁺ ionophore-activated eggs, eggs were collected at 13 or 17 h post-hCG and loaded with fura-2-AM as described above and then washed through three drops of Ca²⁺ /Mg²⁺-free medium (109.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 0.05% PVA). About 15–20 eggs were transferred to a 12 µl drop of Ca²⁺ /Mg²⁺-free Whitten’s medium under light mineral oil in a Leiden chamber and placed on a constant temperature stage on an inverted microscope as above. Immediately prior to imaging, 4 µl of a 40 µM A23187 [GenBank] solution was added to the drop to bring the final concentration of A23187 [GenBank] to 10 µM.

Eggs were illuminated with a 100 W xenon arc lamp; light output was passed through a Lambda 10-2 filter wheel (Sutter Instrument Co., Novato, CA, USA) to alternate excitation wavelengths between 340 and 380 nm. Emitted light passed through a fura-2 bandpass filter cube and was recorded with a Princeton Instruments MicroMAX CCD camera (Roper Scientific, Trenton, NJ, USA). The 340/380 emission ratios were obtained every 10 s. These data were collected for each egg in the field of view and then were analyzed using MetaFluor software (Universal Imaging Corp., West Chester, PA, USA) to assess alterations in whole-egg intracellular Ca²⁺ concentration.

Cortical granule labeling and quantification
Eggs were fixed in 3.7% paraformaldehyde in PBS for 30 min, permeabilized in 0.1% Triton X-100, and blocked in Lens culinaris agglutinin (LCA) block (3 mg/ml NaIO₄ – treated casein, 0.1 M glycine in PBS + 0.02% NaN₃). Intracellular CGs were stained with LCA–biotin (10 µg/ml, Sigma) followed by avidin–FITC (2 µg/ml, Molecular Probes). Eggs were then mounted in 4 µl Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) to flatten them between the slide and coverslip; mounting medium also contained 1.5 µg/ml DAPI to stain DNA. Eggs were imaged using a Nikon S Fluor 100 x oil objective, N.A. 0.5–1.3 (total magnification including 10 x ocular: 1000x), and CG density in the egg cortex was quantified by computer-assisted image quantification using either MetaMorph software (Universal Imaging Corp., West Chester, PA, USA) by counting CG density in measuring four separate ~184 µm² areas in each egg as previously described (Xu et al. 2003) or using IPLabs software (Scanalytics Inc., Fairfax, VA, USA) by counting a 50 µm² by hand and by computer-assisted image quantification, and a 300 µm² area by computer-assisted image quantification as previously described (Abbott et al. 1999, Wortzman & Evans 2005).

Parthenogenetic activation and subsequent challenge with sperm
For experiments with the calcium ionophore A23187 [GenBank] , metaphase II eggs were collected as described above at 13 or 17 h post-hCG. After the 60 min recovery from ZP removal, eggs were washed through three 100 µl drops of Ca²⁺ /Mg²⁺-free media, incubated in a 25 µl drop of Ca²⁺ /Mg²⁺-free Whitten’s medium containing 5 µM A23187 [GenBank] for 5 min, then washed through five 100 µl drops of Ca²⁺ /Mg²⁺-free Whitten’s medium. Activation treatment with A23187 [GenBank] occurred at 15 or 19 h post-hCG for eggs collected at 13 and 17 h post-hCG respectively. As controls for these experiments, eggs were either left unactivated or were activated by fertilization. For sperm-induced activation, ZP-free eggs were inseminated with a sperm:egg ratio of 50:1 (10 eggs per 10 µl drop with 50 000 sperm/ml) for 45 min, then washed through three 100 µl drops of Whitten’s medium containing 15 mg/ml BSA to remove loosely attached sperm. These conditions were optimized to generate monospermic eggs since polyspermic eggs show a higher frequency of Ca²⁺ transients (Faure et al. 1999) and this altered Ca²⁺ signaling could potentially affect egg activation events, including the membrane block to polyspermy.

Eggs were cultured for 3 h following activation by treatment with A23187 [GenBank] or by fertilization, as previous data suggest the membrane block is established in 0.75–2 h post-insemination (Wolf 1978, Horvath et al. 1993, Sengoku et al. 1995, Redkar & Olds-Clarke 1999, McAvey et al. 2002). Previous work had allowed 40 min after ionophore treatment before challenging the eggs with sperm (Wolf et al. 1979). The 3-h incubation used here also allowed the sperm DNA in the fertilized eggs to progress to pronuclear stage so that this sperm could be distinguished from any fertilizing sperm from the second insemination (Wortzman & Evans 2005). After this incubation, eggs in all groups were inseminated for 45 min with a sperm:egg ratio of 50–100:1 (10 eggs added to a 10 µl drop containing 50 000 or 100 000 sperm/ml, freshly collected and capacitated), then washed through three 100 µl drops of Whitten’s medium with 15 mg/ml BSA and fixed in 3.7% paraformaldehyde. Eggs were scored for the number of sperm fused per egg from the second insemination, which could easily be distinguished from the pronuclear stage sperm from the first insemination (Wortzman & Evans 2005).

For experiments with A23187 [GenBank] in which CG exocytosis and cell cycle resumption were assessed, eggs were cultured for 4-h post-activation treatment or insemination, then fixed and stained with DAPI to assess cell cycle resumption and with LCA to stain CGs (described in more detail below; eggs for these analyses were not subjected to the challenge with a second batch of sperm).

Reinsemination assay – challenging zygotes with a second batch of sperm
The design of the reinsemination assay was modified from Wortzman & Evans (2005). The first insemination of the reinsemination experiment (IVF1) was optimized to generate monospermic eggs. The sperm:egg ratios for IVF1 that produced primarily monospermic eggs for each of the experimental groups were as follows: control eggs, 100:1 for 20 min; 1 µM BAPTA-AM-treated eggs, 50:1 for 20 min; 10 µM BAPTA-AM-treated eggs, 30:1 for 20 min. After the first insemination, eggs were washed through three drops of Whitten’s medium containing 15 mg/ml BSA using a thin bore pipette to detach any loosely attached sperm. The fertilized eggs were incubated for 45 or 90 min in a sperm-free drop to allow time for membrane block establishment. After this incubation, eggs were challenged with a second round of insemination (1 h with a sperm:egg ratio of 250:1). Sperm for this second insemination were freshly collected, capacitated for 2–3 h, and labeled with the mitochondrial marker MitoTracker Green (Molecular Probes, Carlsbad, CA, USA). Fifteen minutes before the start of the second insemination, 1 x 10⁶ sperm/ml were incubated with 100 nM MitoTracker Green (diluted from a 1 mM stock in DMSO) for 10 min in the dark at 5% CO₂ in atmosphere; this resulted in 95–100% of the sperm being labeled, and this labeling was not lost in ethanol- or aldehyde-fixed samples (data not shown). After labeling, the sperm were diluted to 250 000 sperm/ml with Whitten’s medium containing 15 mg/ml BSA. As a control, unfertilized eggs were inseminated in parallel with the fertilized egg groups to assess the baseline level of the average number of sperm fused per egg from the second insemination (IVF2 control eggs). Eggs were washed through three drops of Whitten’s medium containing 15 mg/ml BSA using a thin bore pipette to detach any loosely attached sperm. Eggs were fixed in 3.7% paraformaldehyde in PBS and stained with DAPI to assess the stage of maternal DNA and visualize decondensing sperm heads in the egg cytoplasm. Decondensing sperm heads with no detectable green fluorescence in the midpiece (where mitochondria are localized in the sperm) were from the first insemination, and decondensing sperm heads with an associated MitoTracker Green-labeled midpiece were from the second insemination.

Statistical analyses
Statistical analyses (ANOVA with Fishers protected least significant difference post-hoc testing or ² analyses, as indicated) were performed using Statview 5.0 (SAS Institute, Cary, NC, USA). P < 0.05 was considered significant unless otherwise noted. Error bars in figures represent the S.E.M.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The effects of BAPTA-AM treatment on sperm-induced Ca²⁺ transients
We treated eggs with a range of concentrations of BAPTA-AM, as previously described by Kline & Kline (1992), to attenuate sperm-induced Ca²⁺ transients. We focused on 0.5–5 µM BAPTA-AM, based on preliminary experiments that revealed that eggs treated with BAPTA-AM concentrations in this range showed greater extents of polyspermy when compared with untreated control eggs (data not shown). Ca²⁺ imaging experiments were performed on ZP-free eggs treated with 0, 0.5, 1, 2, 5, or 10 µM BAPTA-AM in order to characterize how these BAPTA-AM treatments affected sperm-induced Ca²⁺ transients. A representative Ca²⁺ trace from an egg in each treatment group is shown in Fig. 1, and Table 1 summarizes data on six characteristics of increases in [Ca²⁺]_cyt in all eggs analyzed: the percentage of eggs that had 0, 1, 2, or 3+ transients; the average number of transients in 60 min after the start of insemination; the average amplitude of the first [Ca²⁺]_cyt increase; the average amplitude of subsequent transients (as applicable); the duration of the first [Ca²⁺]_cyt increase (i.e. total time from the start of the increase to the time when [Ca²⁺]_cyt returned to basal levels); and the time from the start of the insemination to the start of the increase in [Ca²⁺]_cyt. We observed mild attenuation of sperm-induced Ca²⁺ transients with low BAPTA-AM concentrations (0.5, 1 µM) and more severe attenuation at higher concentrations (2, 5, 10 µM). Eggs treated with 0.5 or 1 µM BAPTA-AM typically had one [Ca²⁺]_cyt increase that was lower in amplitude and longer in duration than the first transient in control eggs. All of the eggs treated with 0.5 or 1 µM BAPTA-AM experienced some increase in [Ca²⁺]_cyt, whereas only 79% of eggs treated with 2 µM BAPTA-AM experienced an increase in [Ca²⁺]_cyt. The increases in [Ca²⁺]_cyt in the eggs in this 2 µM group were lower in amplitude and longer in duration than the first transient in control eggs; these decreases in amplitude and increases in duration were more extensive than those observed in eggs treated with 0.5 or 1 µM BAPTA-AM. Eggs treated with 5 µM BAPTA-AM were distinguished from the other groups, in that only two of 24 eggs imaged showed an increase in [Ca²⁺]_cyt. We verified that the eggs in the 5 µM BAPTA-AM treatment group were fertilized by staining them with DAPI to visualize decondensing sperm heads in the egg cytoplasm (data not shown). All the post-fertilization Ca²⁺ transients were completely abolished in eggs treated with 10 µM BAPTA-AM in our experiments, in agreement with previous studies (Kline & Kline 1992).

View larger version (22K): [in this window] [in a new window]   Figure 1 Representative traces of [Ca2+]cyt transients in fertilized BAPTA-AM-treated eggs. ZP-free eggs were loaded with fura 2-AM for 20 min, then loaded with 0.5, 1, 2, 5, or 10 µM BAPTA-AM for 60 min prior to insemination. Control eggs were incubated in medium containing 0.1% DMSO. The sperm were added at the start of imaging shown on each tracing (t = 0). Fertilization-induced Ca2+ transients were detected by exciting fura-2 fluorescence and measuring the intensity of light emitted (340/380 nm). A shows a representative [Ca2+]cyt profile for a control egg (DMSO-treated, 0 µM BAPTA-AM). B–F show representative [Ca2+]cyt profiles for eggs treated with 0.5, 1, 2, 5, or 10 µM BAPTA-AM respectively.

View larger version (19K): [in this window] [in a new window]   Figure 2 Effect of attenuated Ca2+ transients on the extent of polyspermy. ZP-free eggs were incubated in either DMSO or 0.5, 1, 2, or 5 µM BAPTA-AM for 60 min prior to insemination and washed free of BAPTA-AM before fertilization. Eggs were inseminated with a sperm:egg ratio of 50:1, washed free of loosely bound sperm, and fixed to determine the extent of sperm–egg fusion. A shows the average number of sperm fused per egg when the eggs were inseminated for 45 min or 1.5 h; data are based on three experiments with 27–41 eggs per group per timepoint. Except for the 0 µM treatment group, the increase in the extent of polyspermy from 0.75 to 1.5 h within each concentration group is statistically significant (P < 0.01). B shows the average number of sperm fused per egg when the eggs were inseminated for 1.5 or 4 h; data are based on four experiments with 41–64 eggs per group per timepoint. Only the 0 µM treatment group had a statistically significant increase in the extent of polyspermy from 1.5 to 4 h (P = 0.04). All treatment groups are significantly different from one another within a given timepoint (P < 0.05) with the following exceptions: at 0.75 h post-insemination, the difference between the 0 and 0.5 µM groups is not significant; and at 4 h post-insemination, the difference between the 1 and 2 µM groups is not significant. Data in A and B are from experiments with both timepoints performed in parallel on the same day. Error bars represent the S.E.M.

Effects of calcium ionophore-induced [Ca²⁺]_cyt increase on membrane block establishment
The data in Fig. 2 indicated that the membrane block to polyspermy was not an ‘all or none’ response by the egg; as sperm-induced Ca²⁺ transients were increasingly attenuated, the more sperm were fused with the eggs. Additionally, eggs treated with 0.5 µM BAPTA-AM were only slightly more polyspermic than control untreated eggs, but most of these eggs only experienced one transient increase in [Ca²⁺]_cyt (Fig. 1B, Table 1). This observation raised the question of whether a single increase in [Ca²⁺]_cyt was sufficient to establish a partially effective membrane block to polyspermy, and prompted us to examine the effects of parthenogenetic activation with the Ca²⁺ ionophore A23187 [GenBank] , which induces a single increase in [Ca²⁺]_cyt, on membrane block establishment. While it has been reported previously that parthenogenetically activated eggs did not establish a membrane block to polyspermy, our analyses here included some important variables not included in previous studies (Wolf et al. 1979, Horvath et al. 1993). First, we tested the hypothesis that postovulatory time could affect the ability of eggs to establish a membrane block to polyspermy in response to treatment with Ca²⁺ ionophore, as aged eggs are more responsive to parthenogenetic stimuli (Fulton & Whittingham 1978, Nagai 1987, Collas et al. 1989, Ware et al. 1989, Fissore & Robl 1992, Abbott et al. 1998). Previous studies of membrane block establishment in response to ionophore treatment only tested freshly ovulated eggs (Wolf et al. 1979). Secondly, we tested the hypothesis that Ca²⁺ ionophore may induce a partially effective membrane block. Our analyses here included eggs activated by a fertilizing sperm; this control had been omitted from previous studies but was crucial to include here in light of our data that membrane blocks may vary in effectiveness (e.g. in eggs experiencing different levels of attenuated Ca²⁺ transients; Fig. 2).

Ca²⁺ imaging showed that A23187 [GenBank] treatment, induced a single transient increase in [Ca²⁺]_cyt in eggs collected at 13 and 17 h after hCG injection (Table 2), as previously reported (Kline & Kline 1992, Swann & Ozil 1994, Jellerette et al. 2000; note: we did not test eggs at > 17 h post-hCG since we have demonstrated that eggs collected at 22 h post-hCG have a reduced ability to be fertilized and to establish a membrane block (Wortzman & Evans 2005). To determine how an A23187 [GenBank] -induced increase in [Ca²⁺]_cyt impacted downstream egg activation responses, eggs treated with 5 µM A23187 [GenBank] were compared with unfertilized/unactivated eggs and eggs activated by sperm, using insemination conditions optimized to obtain monospermic eggs. Activated eggs were cultured for 3 h and then challenged with sperm. As controls, CG exocytosis and cell cycle resumption were analyzed 4 h after activation treatment or insemination in a subset of eggs from each group.

View larger version (21K): [in this window] [in a new window]   Figure 3 Ability of A23817-treated eggs to undergo CG exocytosis, resume the cell cycle, and establish a membrane block to polyspermy. Eggs were collected 13 or 17 h post-hCG, then activated with either 5 µM A23187 or sperm (as detailed in the Materials and Methods), or left unactivated/unfertilized. A–C show data from eggs collected 13 h post-hCG, and D–F show data from eggs collected 17 h post-hCG. A and D show CG densities in unactivated control eggs and in eggs activated with A23187 or sperm (eggs were fixed at 4 h post-activation). Data are expressed as CGs/100 µm2 (n = 27–47 eggs for each treatment group from four experiments). B and E show what percentages of A23187-treated or inseminated eggs had exited from metaphase II arrest by 4 h post-treatment. Note: inseminated eggs include a small number of eggs that were not fertilized and thus remained arrested at metaphase II. C and F show the average number of sperm fused per egg after insemination with a sperm:egg ratio of 50:1 for 45 min in unactivated/unfertilized eggs, A23187-treated eggs or previously fertilized eggs (n = 32–53 eggs for each treatment group from three experiments; note: values in C and F for eggs activated by sperm represent only decondensing sperm heads, excluding pronuclear stage sperm from the first insemination.)

To determine which of these deficiencies existed in Ca²⁺-suppressed eggs, we used a ‘reinsemination’ assay, in, which fertilized ZP-free eggs were challenged with a second batch of sperm and assessed for how many sperm from the second insemination were able to penetrate the eggs after either 45- or 90-min recovery period following the first insemination. Our controls were: (1) untreated fertilized eggs that experienced normal sperm-induced Ca²⁺ signaling; and (2) unfertilized ‘naïve’ eggs that were only challenged with sperm in parallel with the second insemination of the fertilized eggs. These controls were compared with eggs treated with 1 µM BAPTA-AM (one sperm-induced Ca²⁺ transient) or 10 µM BAPTA-AM (completely abolished sperm-induced Ca²⁺ transients; Fig. 1, Table 1).

When reinseminated after 45 min, both groups of Ca²⁺-suppressed eggs (1 or 10 µM BAPTA-AM) became fertilized to the same extent as naïve, unfertilized control eggs (IVF2 control; Fig. 4A), demonstrating that the Ca²⁺-suppressed eggs had not established an effective membrane block at this time. However, when reinseminated after a 90 min recovery time, Ca²⁺-suppressed eggs were as effective as control fertilized eggs in preventing additional sperm fusion (Fig. 4A). This finding suggested that the post-fertilization Ca²⁺ transients played a role in the proper timing of membrane block establishment, as the membrane block was established more slowly in Ca²⁺-suppressed eggs. Additionally, Ca²⁺ transients were not solely responsible for membrane block establishment because eggs that had no post-fertilization Ca²⁺ transients were still able to prevent additional sperm fusion upon reinsemination, albeit more slowly.

View larger version (19K): [in this window] [in a new window]   Figure 4 Ability of Ca2+-suppressed eggs to prevent additional fertilization upon reinsemination. A shows the average number of sperm fused per egg from the second insemination (average number of MitoTracker green-labeled sperm per egg) in unfertilized eggs and fertilized eggs treated with 0–10 µM BAPTA-AM. Data are based on 39–75 eggs per treatment group from four experiments. B shows the average number of MitoTracker green-labeled sperm fused per egg in the subset of eggs from A that were monospermic after IVF1 (69–87% of the total group). C shows the average number of MitoTracker green-labeled sperm fused per egg in the subset of eggs from A that were polyspermic after IVF1 (13–31% of the total group). Data are from experiments with both timepoints performed in parallel on the same day. A statistically significant difference between unfertilized control levels (IVF2) and a treatment group is represented by an a (P < 0.05), and a statistically significant difference between monospermic and polyspermic eggs from IVF1 within a treatment group is represented by a b (P < 0.012). Error bars represent the S.E.M.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We have previously shown that complete suppression of [Ca²⁺]_cyt transients in fertilized ZP-free mouse eggs, leads to an increase in the number of fused sperm (McAvey et al. 2002), indicative of disruption in the ability of the eggs to establishment a membrane block to prevent polyspermy. Here, we extend those findings by defining the effects of partial suppression of sperm-induced Ca²⁺ transients on the membrane block, by determining that Ca²⁺ plays a role in the timing of membrane block establishment, and by documenting that the is a Ca²⁺-independent component of the pathway leading to membrane block establishment.

Mouse eggs that experience only one sperm-induced Ca²⁺ transient establish a membrane block that is less effective at preventing multiple fertilizations than the membrane block in eggs that experience normal sperm-induced Ca²⁺ transients (Fig. 2). However, the membrane block in these eggs that experience only one sperm-induced Ca²⁺ transient is substantially more effective than the block in eggs treated with 5 or 10 µM BAPTA-AM to suppress Ca²⁺ transients completely, indicating that one sperm-induced transient increase in [Ca²⁺]_cyt can trigger the egg to establish a membrane block that is intermediate in effectiveness. This discovery is somewhat surprising since it had been previously reported that Ca²⁺ ionophore, which induces only one transient increase in [Ca²⁺]_cyt, does not induce membrane block establishment (Wolf et al. 1979). This prompted us to reexamine the effects of Ca²⁺ ionophore treatment on membrane block establishment. Our studies here, include the important variable of post-ovulatory aged eggs (omitted from previous membrane block studies) because aged eggs are more responsive to parthenogenetic stimuli (Fulton & Whittingham 1978, Nagai 1987, Collas et al. 1989, Ware et al. 1989, Fissore & Robl 1992, Abbott et al. 1998). We find that neither freshly ovulated nor post-ovulatory aged eggs establish even a partially effective membrane block in response to ionophore treatment, despite the fact that the Ca²⁺ ionophore-induced transient in [Ca²⁺]_cyt has similar characteristics to the transient in eggs treated with 0.5 and 1.0 µM BAPTA-AM. These data provide evidence that some aspect of sperm-induced Ca²⁺ transients is required for membrane block establishment and/or that some event associated with fertilization (and lacking in parthenogenetic activation) is part of the pathway leading to membrane block establishment.

Additional insights into these issues come from our work here that defines why eggs with little or no sperm-induced [Ca²⁺]_cyt increase following fertilization become more polyspermic than control eggs. Our studies show that Ca²⁺ regulates the timing of membrane block establishment. The membrane block is established more slowly in eggs with attenuated or completely suppressed Ca²⁺ transients than in control eggs that experience normal sperm-induced Ca²⁺ transients, and thus additional sperm have a larger time window in which to fertilize an egg after the first sperm has fused with the egg. These studies also reveal that polyspermic zygotes have fewer MitoTracker-labeled sperm from the second insemination (IVF2) fused with them than do the monospermic eggs. Interestingly, this was observed for control untreated eggs (experiencing normal sperm-induced Ca²⁺ signaling) and eggs treated with 1 or 10 µM BAPTA-AM (Fig. 4B and C). These results indicate that: (a) polyspermic zygotes establish a more effective membrane block and were better able to prevent additional fertilization events than were monospermic zygotes; and (b) the effect on the membrane block of multiple sperm–egg interaction events resulting from polyspermy is independent of increased [Ca²⁺]_cyt in the egg. Taken together, these data complement the conclusions from the studies noted above and provide evidence that a fertilization-associated event(s) is a part of the pathway leading to membrane block establishment, with Ca²⁺ playing a role to facilitate membrane block establishment.

This study demonstrates that the membrane block differs from other post-fertilization egg activation responses as Ca²⁺ plays a role in facilitating membrane block establishment but is not the only stimulus. The additional stimulus/stimuli appears to be associated with some event(s) occurring with fertilization. The role of Ca²⁺ in regulation of the timing of membrane block establishment is clearly important, since the assays of sperm incorporation over time (Fig. 2) show that an increased extent of polyspermy can be associated with this delay. An optimal, sperm-induced pathway of Ca²⁺ signaling may be important, as it is known that spatial and temporal characteristics of Ca²⁺ transients differ between eggs activated by IVF and eggs activated by parthenogenetic stimuli and also eggs fertilized by intracytoplasmic sperm injection (Nakano et al. 1997, Sato et al. 1999, Deguchi et al. 2000). ICSI is pertinent here as it too, like parthenogenesis, has been reported to be unable to induce establishment of the membrane block (Maleszewski et al. 1996). Several cellular and molecular events occur with fertilization and are different or lacking in eggs activated parthenogenetically or by ICSI. These include sperm–egg binding, sperm–egg fusion, and sperm-induced changes in the egg cortex and cytoplasm, and any of these may also function with the aforementioned spatial–temporal characteristics of Ca²⁺ transients to effect membrane block establishment. The membrane block to polyspermy in mammalian eggs is likely to be the culmination of multiple events that together modify the egg membrane’s receptivity to sperm.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This work was supported by grants from the NIH/NICHD (HD045671, HD037696) and the March of Dimes (6-FY-04-59) to J P E, and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences to C J W. A J G has been supported by a training grant from the NIH (HD07276). We are grateful to Dr Karl Broman (Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health) and Matthew Marcello (lab of J P E) for assistance with statistical analyses, and to members of the Evans lab for discussions of the data. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 31 October 2006
Accepted 30 November 2006

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

Abbott AL & Ducibella T 2001 Calcium and the control of mammalian cortical granule exocytosis. Frontiers in Bioscience 6 D792–D806.[Web of Science][Medline]

Abbott AL, Xu Z, Kopf GS, Ducibella T & Schultz RM 1998 In vitro culture retards spontaneous activation of cell cycle progression and cortical granule exocytosis that normally occur in in vivo unfertilized mouse eggs. Biology of Reproduction 59 1515–1521.[Abstract/Free Full Text]

Abbott AL, Fissore RA & Ducibella T 1999 Incompetence of preovulatory mouse oocytes to undergo cortical granule exocytosis following induced calcium transients. Developmental Biology 207 38–48.[CrossRef][Web of Science][Medline]

Austin CR 1961 The Mammalian Egg, Springfield, IL: Charles C. Thomas.

Berridge MJ, Bootman MD & Roderick HL 2003 Calcium signalling: dynamics, homeostasis and remodelling. Nature Review. Molecular and Cellular Biology 4 517–529.

Collas P, Balise JJ, Hofmann GA & Robl JM 1989 Electrical activation of mouse oocytes. Theriogenology 32 835–844.[CrossRef][Web of Science][Medline]

Deguchi R, Shirakawa H, Oda S, Mohri T & Miyazaki S 2000 Spatiotemporal analysis of Ca²⁺ waves in relation to the sperm entry site and animal-vegetal axis during Ca²⁺ oscillations in fertilized mouse eggs. Developmental Biology 218 299–313.[CrossRef][Web of Science][Medline]

Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz RM, Kopf GS, Fissore R, Madoux S & Ozil JP 2002 Egg-to-embryo transition is driven by differential responses to Ca²⁺ oscillation number. Developmental Biology 250 280–291.[CrossRef][Web of Science][Medline]

Evans JP, Schultz RM & Kopf GS 1995 Mouse sperm-egg membrane interactions: analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) b. Journal of Cell Science 108 3267–3278.[Abstract]

Faure JE, Myles DG & Primakoff P 1999 The frequency of calcium oscillations in mouse eggs at fertilization is modulated by the number of fused sperm. Developmental Biology 213 370–377.[CrossRef][Web of Science][Medline]

Fissore RA & Robl JM 1992 Intracellular Ca²⁺ response of rabbit oocytes to electrical stimulation. Molecular Reproduction and Development 32 9–16.[CrossRef][Web of Science][Medline]

Fulton BP & Whittingham DG 1978 Activation of mammalian oocytes by intracellular injection of calcium. Nature 273 149–151.[CrossRef][Medline]

Gardner AJ & Evans JP 2006 Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm. Reproduction, Fertility, and Development 18 53–61.[CrossRef][Medline]

Gould MC & Stephano JL 2003 Polyspermy prevention in marine invertebrates. Microscopy Research and Technique 61 379–388.[CrossRef][Web of Science][Medline]

Horvath PM, Kellom T, Caulfield J & Boldt J 1993 Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs. Molecular Reproduction and Development 34 65–72.[CrossRef][Web of Science][Medline]

Hyslop LA, Nixon VL, Levasseur M, Chapman F, Chiba K, McDougall A, Venables JP, Elliott DJ & Jones KT 2004 Ca²⁺-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest. Developmental Biology 269 206–219.[CrossRef][Web of Science][Medline]

Igusa Y, Miyazaki S & Yamashita N 1983 Periodic increase in cytoplasmic Ca²⁺ reflected in hyperpolarizing responses of the egg during cross-species fertilization between hamster and mouse. Journal of Physiology 340 633–647.[Abstract/Free Full Text]

Jaffe LA & Gould M 1985 Polyspermy-preventing mechanisms. In Biology of Fertilization: The Fertilization Response of the Egg, pp 223–250. Eds CB Metz & A Monroy. Orlando, FL: Academic Press.

Jaffe LA, Sharp AP & Wolf DP 1983 Absence of an electrical polyspermy block in the mouse. Developmental Biology 96 317–323.[CrossRef][Web of Science][Medline]

Jellerette T, He CL, Wu H, Parys JB & Fissore RA 2000 Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation. Developmental Biology 223 238–250.[CrossRef][Web of Science][Medline]

Kline D & Kline JT 1992 Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Developmental Biology 149 80–89.[CrossRef][Web of Science][Medline]

Kubiak JZ 1989 Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest. Developmental Biology 136 537–545.[CrossRef][Web of Science][Medline]

Malcuit C, Kurokawa M & Fissore RA 2006 Calcium oscillations and mammalian egg activation. Journal of Celluar Physiology 206 565–573.

Maleszewski M, Kimura Y & Yanagimachi R 1996 Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: evidence based on intracytoplasmic sperm injection experiments in the mouse. Molecular Reproduction and Development 44 256–259.[CrossRef][Web of Science][Medline]

Maluchnik D & Borsuk E 1994 Sperm entry into fertilised mouse eggs. Zygote 2 129–131.[Medline]

McAvey BA, Wortzman GB, Williams CJ & Evans JP 2002 Involvement of calcium signaling and the actin cytoskeleton in the membrane block to polyspermy in mouse eggs. Biological Reproduction 67 1342–1352.

McCulloh DH, Rexroad CE Jr & Levitan H 1983 Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials. Developmental Biology 95 372–377.[CrossRef][Web of Science][Medline]

Miyazaki S & Igusa Y 1981 Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations. Nature 290 702–704.[CrossRef][Medline]

Nagai T 1987 Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol. Gamete Research 16 243–249.[CrossRef][Web of Science][Medline]

Nakano Y, Shirakawa H, Mitsuhashi N, Kuwabara Y & Miyazaki S 1997 Spatiotemporal dynamics of intracellular calcium in the mouse egg injected with a spermatozoon. Molecular Human Reproduction 3 1087–1093.[Abstract/Free Full Text]

Ozil JP, Markoulaki S, Toth S, Matson S, Banrezes B, Knott JG, Schultz RM, Huneau D & Ducibella T 2005 Egg activation events are regulated by the duration of a sustained [Ca²⁺]_cyt signal in the mouse. Developmental Biology 282 39–54.[CrossRef][Web of Science][Medline]

Redkar AA & Olds-Clarke PJ 1999 An improved mouse sperm-oocyte plasmalemma binding assay: Studies on characteristics of sperm binding in medium with or without glucose. Journal of Andrology 20 500–508.[Abstract/Free Full Text]

Runft LL, Jaffe LA & Mehlmann LM 2002 Egg activation at fertilization: where it all begins. Developmental Biology 245 237–254.[CrossRef][Web of Science][Medline]

Sato MS, Yoshitomo M, Mohri T & Miyazaki S 1999 Spatiotemporal analysis of [Ca²⁺]_i rises in mouse eggs after intracytoplasmic sperm injection (ICSI). Cell Calcium 26 49–58.[CrossRef][Web of Science][Medline]

Sengoku K, Tamate K, Horikawa M, Takaoka Y, Ishikawa M & Dukelow WR 1995 Plasma membrane block to polyspermy in human oocytes and preimplantation embryos. Journal of Reproduction Fertility 105 85–90.

Swann K & Ozil J-P 1994 Dynamics of the calcium signal that triggers mammalian egg activation. International Review of Cytology 152 183–222.[Web of Science][Medline]

Ware CB, Barnes FL, Maiki-Laurila M & First NL 1989 Age dependence of bovine oocyte activation. Gamete Research 22 265–275.[CrossRef][Web of Science][Medline]

Whitten WK 1971 Nutrient requirements for the culture of preimplantation embryos in vitro. Advances in the Bio Sciences 6 129–139.

Wolf DP 1978 The block to sperm penetration in zona-free mouse eggs. Developmental Biology 64 1–10.[CrossRef][Web of Science][Medline]

Wolf DP, Nicosia SV & Hamada M 1979 Premature cortical granule loss does not prevent sperm penetration of mouse eggs. Developmental Biology 71 22–32.[CrossRef][Web of Science][Medline]

Wortzman GB & Evans JP 2005 Membrane and cortical abnormalities in post-ovulatory aged eggs: analysis of fertilizability and establishment of the membrane block to polyspermy. Molecular Human Reproduction 11 1–9.[Abstract/Free Full Text]

Xu Z, Williams CJ, Kopf GS & Schultz RM 2003 Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca²⁺ oscillatory behavior in mouse eggs. Developmental Biology 254 163–171.[CrossRef][Web of Science][Medline]

Yanagimachi R 1994 Mammalian fertilization. In The Physiology of Reproduction, 2 edn, pp 189–317. Eds E Knobil & JD Neill. New York: Raven Press, Ltd..

Zuccotti M, Yanagimachi R & Yanagimachi H 1991 The ability of hamster oolemma to fuse with spermatozoa: Its acquisition during oogenesis and loss after fertilization. Development 112 143–152.[Abstract]

This article has been cited by other articles:

				U. Vjugina, X. Zhu, E. Oh, N. J. Bracero, and J. P. Evans Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion Biol Reprod, April 1, 2009; 80(4): 833 - 841. [Abstract] [Full Text] [PDF]

				G. B. Wortzman-Show, M. Kurokawa, R. A. Fissore, and J. P. Evans Calcium and sperm components in the establishment of the membrane block to polyspermy: studies of ICSI and activation with sperm factor Mol. Hum. Reprod., August 1, 2007; 13(8): 557 - 565. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplementary Figure Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gardner, A. J Articles by Evans, J. P Search for Related Content PubMed PubMed Citation Articles by Gardner, A. J Articles by Evans, J. P