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.
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
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.
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 × 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 solution was added to the drop to bring the final concentration of A23187 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 × oil objective, N.A. 0.5–1.3 (total magnification including 10 × ocular: 1000×), 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, 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 for 5 min, then washed through five 100 μl drops of Ca2+ /Mg2+-free Whitten’s medium. Activation treatment with A23187 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 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 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 × 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 (ANOVA with Fishers protected least significant difference post-hoc testing or χ2 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.
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).
The effects of attenuated sperm-induced Ca2+ transients on the membrane block to polyspermy and other egg activation events
We next determined how attenuation of sperm-induced Ca2+ transients affected the establishment of the membrane block to polyspermy by examining the average number of sperm fused per egg over time in ZP-free eggs treated with 0, 0.5, 1, 2, or 5 μM BAPTA-AM. The number of sperm fused per ZP-free egg normally plateaus with increased time post-insemination, indicative of the establishment of a membrane block to polyspermy that prevents additional sperm from fusing with fertilized eggs (Wolf 1978, Sengoku et al. 1995, Redkar & Olds-Clarke 1999, McAvey et al. 2002). One experimental series examined sperm–egg fusion at 0.75 and 1.5 h post-insemination (Fig. 2A), and the second examined sperm–egg fusion at 1.5 and 4 h post-insemination (Fig. 2B). Increased attenuation of the post-fertilization Ca2+ transients with increasing concentrations of BAPTA-AM resulted in increased polyspermy of ZP-free eggs. In addition, the average number of sperm fused per egg increased significantly from 0.75 to 1.5 h post-insemination in eggs treated with 0.5, 1, 2, or 5 μM BAPTA-AM (P < 0.01) but not in control eggs. The average number of sperm fused per egg at each post-insemination timepoint was different between virtually all BAPTA-AM treatment groups when compared with untreated controls (figure legend details the statistical comparisons). These data indicate that eggs with increasingly attenuated sperm-induced Ca2+ transients establish less effective membrane blocks to polyspermy.
As a control and in order to understand completely the effects of BAPTA-AM-mediated attenuation of sperm-induced Ca2+ transients on egg activation, we also examined cell cycle resumption and CG exocytosis (Supplementary Figure 1, which can be viewed online at http://www.reproduction-online.org/supplemental/). Three different cell cycle resumption endpoints were assessed, as was done previously (Ducibella et al. 2002): (1) metaphase II arrest, (2) full progression to embryonic interphase, and (3) metaphase III which is an intermediate state in which the egg completes meiosis but does not continue to embryonic interphase (as a result of M-phase promoting factor (MPF) activity decreasing only transiently, due to insufficient [Ca2+]cyt increases, and then returning to high levels (Kubiak 1989, Ducibella et al. 2002, Hyslop et al. 2004)). As the post-fertilization Ca2+ transients were increasingly attenuated, the proportion of eggs exiting metaphase II arrest gradually decreased, while the proportion of eggs remaining arrested at metaphase II increased (Supplementary Figure 1, which can be viewed online at http://www.reproduction-online.org/supplemental/). A subset of eggs (4–23%) in the 0.5–5 μM treatment groups resumed the cell cycle but regressed to metaphase III. BAPTA-AM attenuation of sperm-induced Ca2+ transients affected the kinetics as well as the overall extent of CG exocytosis (supplemental data). In eggs treated with 0.5 or 1 μM BAPTA-AM, CG exocytosis was slightly reduced at 0.75 h post-insemination but reached control levels by 4 h post-insemination. In eggs treated with 2 μM BAPTA-AM, both the timing and the extent of CG exocytosis were affected. Eggs treated with 5 μM BAPTA-AM had the overall extent of CG exocytosis reduced by ~20% at both 0.75 and 4 h post-insemination when compared with pre-insemination CG densities. Taken together, these data on eggs experiencing attenuated sperm-induced Ca2+ transients, like the data from electropermeabilization studies (Ducibella et al. 2002), support the conclusion that there are distinct Ca2+ requirements for the initiation and the completion of cell cycle resumption and CG exocytosis.
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, 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 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-induced increase in [Ca2+]cyt impacted downstream egg activation responses, eggs treated with 5 μM A23187 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.
None of the eggs treated with A23187 appeared to establish a membrane block to polyspermy, regardless of whether the eggs were collected 13 or 17 h post-hCG. The number of sperm fused per egg was similar for unfertilized eggs and A23187-treated eggs, whereas virtually no sperm fused with eggs that were activated by fertilization (Fig. 3C and F). Moderate levels of CG exocytosis and low levels of cell cycle resumption (similar to those observed previously with A23187 (Abbott et al. 1998)) occurred in A21387-treated eggs that were collected at 17 h post-hCG (Fig. 3D and E), demonstrating that A23187 treatment induced egg activation responses, albeit to a lesser extent than fertilization did, in agreement with previous studies. Little or no CG exocytosis or exit from metaphase II arrest occurred in A23187-treated eggs that had been collected at 13 h post-hCG (Fig. 3A and B), in agreement with previous demonstrations of low responsiveness of ‘young’ eggs to parthenogenetic stimuli (Fulton & Whittingham 1978, Nagai 1987, Collas et al. 1989, Ware et al. 1989, Fissore & Robl 1992, Abbott et al. 1998).
Investigating the deficiencies in the membrane blocks of Ca2+-suppressed eggs
As shown above, eggs experiencing attenuated sperm-induced Ca2+ transients establish a less effective membrane block to polyspermy (Fig. 2). We next sought to define more precisely how the membrane blocks of Ca2+-suppressed eggs were deficient in preventing additional fertilization, testing the following possibilities: (1) the membrane block is less robust and therefore less effective at preventing additional sperm fusion; (2) the membrane block is slower to establish and thus additional sperm can 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.
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.
We gained additional insights from comparing the subset of zygotes that were monospermic after IVF1 to the subset of zygotes that were polyspermic after IVF1. Interestingly, polyspermic zygotes had fewer Mito-Tracker-labeled sperm fused with them than did the monospermic eggs. This was observed in zygotes derived from control untreated eggs (experiencing normal sperm-induced Ca2+ transients) and from eggs treated with 1 or 10 μM BAPTA-AM (Fig. 4B and C). There was also moderate evidence for a correlation between the extent of polyspermy from IVF1 and whether eggs were penetrated by sperm from IVF2 (i.e. eggs that were dispermic or trispermic or more from IVF1 were increasingly less likely to contain sperm from IVF2; χ2 analysis P = 0.07 for control eggs, P = 0.01 for eggs treated with 1 μM BAPTA-AM, and P = 0.06 for eggs treated with 10 μM BAPTA-AM).
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.
Characteristics of sperm-induced increases in [Ca2+]cyt in BAPTA-AM-treated eggs.
|Percentage of eggs with a given number of Ca2+ transients in the 60 min from the start of insemination||Average amplitude (340/380)|
|BAPTA-AM (μM)||Number of eggs imaged||0 (%)||1 (%)||2 (%)||3+ (%)||Av. no.of Ca2+transients in the 60 min from the start of insemination||First transient||Subsequent transient||Duration of first transient (min)||Time to first transient (min)|
|aStatistically different from 0 μM. bOnly 1/31 eggs had more than one Ca2+ transient. cStatistically different from 0.5 μM. dStatistically different from 2 μM. eStatistically different from 1 μM. fBased on only 2/24 eggs; n too small for statistical analysis.|
|0||11||0||0||0||100||4.73 ± 0.41||2.58 ± 0.10||1.92 ± 0.07||4.15 ± 0.15||14.63 ± 1.89|
|0.5||31||0||97||3||0||1.07 ± 0.05a||2.18 ± 0.11a||1.2b||8.46 ± 0.39a||23.90 ± 2.17a|
|1||16||0||100||0||0||1.00 ± 0.00a||1.41 ± 0.17a,c||N/A||10.34 ± 0.76a,c,d||16.75 ± 1.45|
|2||14||21||79||0||0||1.00 ± 0.00a||1.14 ± 0.05a,c||N/A||13.79 ± 0.54a,c,e||22.61 ± 4.79|
|5||24||92||8||0||0||0.08 ± 0.06a,c,d,e||0.93 ± 0.08f||N/A||10.45 ± 0.35f||42.05 ± 5.65f|
Characteristics of increases in [Ca2+]cyt after treatment with A23187.
|Number of transients in 60 min of imaging|
|Time of egg collection||Number of eggs analysed||1 (%)||2+ (%)||Average peak amplitude of transient (340/380)||Average duration of transient (min)|
|*Indicates statistically significantly different from the value for the 13 h post-hCG (P < 0.05).|
|13 h Post-hCG||14||100||0||1.85 ± 0.03||8.20 ± 0.42|
|17 h Post-hCG||31||100||0||1.76 ± 0.04||11.16 ± 0.55*|
Received 31 October 2006 Accepted 30 November 2006
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.
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