The passage of Ca²⁺ and fluorescent markers between the sperm and egg after fusion in the mouse

Activation of the egg at fertilization in a wide variety of species is triggered by changes in the intracellular Ca²⁺ concentration (Zucker et al., 1978; Kline 1988; Kline and Kline, 1992; McDougall et al., 1995). However, the mechanism employed by the sperm to trigger Ca²⁺ release remains uncertain (reviewed in Jaffe, 1991; Whitaker and Swann, 1993; Schultz and Kopf, 1995; Swann and Lai, 1997). Three models have been proposed to explain the rise in Ca²⁺ termed the ‘conduit’, ‘contact’ and ‘content’ models (Jaffe, 1991).

The ‘conduit’ model proposes that the sperm acts as a channel to allow Ca²⁺ from the extracellular medium to enter the egg, thereby overloading stores and causing Ca²⁺ release (Jaffe, 1991). Alternatively, sperm may be a source of Ca²⁺, an idea which was first proposed in the ‘Ca²⁺ -bomb’ hypothesis (Jaffe, 1980, 1983) and which would be favoured by the increase in mammalian sperm Ca²⁺ during capacitation and acrosome reaction (Visconti and Kopf, 1998; Florman et al., 1998; for review see Yanagimachi, 1994). In frog and fish eggs, further support for this idea is provided by the observation that a single Ca²⁺ wave is observed at fertilization which is mimicked by surface membrane damage (Gilkey, 1983; Kubota et al., 1987). This is not the case for sea urchin (Swann and Whitaker, 1986) although Ca²⁺ chelators and La³⁺ block egg activation (Creton and Jaffe, 1995) (note that the latter might also be considered supportive of the ‘conduit’ model -see above). In mammalian eggs, there are a series of low frequency transients (Cuthbertson and Cobbold, 1985) which are not produced by Ca²⁺ injection or electroporation (McNiven et al., 1988; Fissore and Robl, 1992; Sun et al., 1992; Collas et al., 1993; Ozil and Swann, 1995). In any case, such interventions may not mimic the behaviour of a slow Ca²⁺ leak from the sperm (Jaffe, 1990; Creton and Jaffe, 1995).

The ‘contact’ model proposes that sperm bind to the plasma membrane to activate phospholipase C (PLC) and produce inositol trisphosphate (IP₃). The involvement of this pathway is suggested by the oscillations produced by IP₃ injection (Swann et al., 1989; Jones and Whittingham, 1996) and the block of sperm-induced oscillations by PLC inhibition (DuPont et al., 1996). Furthermore, an antibody to the IP3 receptor blocks sperm’s ability to release Ca²⁺ (Miyazaki et al., 1992). However, there has been no report of a sperm-derived ligand which interacts with the egg membrane to cause Ca²⁺ oscillations in mammals.

The ‘content’ model proposes that a sperm component (sperm factor) is responsible for Ca²⁺ release in the egg. At present, there are three possible candidates. (1) Glucosamine-6-phosphate deaminase, a protein composed of 33 kDa monomers (Parrington et al., 1996); however the recombinant protein has no Ca²⁺ releasing activity (Wolosker et al., 1998). (2) A truncated form of c-kit (tr-kit), a 23 kDa protein which causes Ca²⁺-dependent egg activation (Sette et al., 1997). (3) Calicin, a 60 kDa protein found in the ‘calyx’ (Kimura et al., 1998). Deaminases have been reported to be hexameric (Calcagno et al., 1984) and tr-kit yields a 48 kDa product (Albanesi et al., 1996). Therefore, current ‘content’ models require the formation of large pores but only 10 kDa dextrans have been shown to pass between egg and sperm before the Ca²⁺ transient occurs (Lawrence et al., 1997).

Using two-photon (TPM) and confocal microscopy we have measured Ca²⁺ changes which occur in mouse eggs around the time of sperm fusion. We show that the pores which form after sperm fusion allow very high molecular weight proteins to pass between egg and sperm – as required by the ‘content’ model. No evidence was found for a role of extracellular Ca²⁺ in initiation of the first Ca²⁺ transient.

4-to 8-week old female F₁ hybrid mice of B6CB (C57Bl/6JLac × CBA/CaLac), were superovulated by serial injection of 7.5 i.u. pregnant mares’ serum gonadotrophin and 5 i.u. human chorionic gonadotrophin (hCG) given 48 hours apart. Eggs were collected 13-14 hours post-hCG injection in medium M2 (Fulton and Whittingham, 1978; Quinn et al., 1982). The cumulus cells were removed by a brief incubation in hyaluronidase (0.3 mg/ml). Sperm were collected from known fertile F₁ hybrid mice and capacitated for at least 2 hours in medium T6 at a concentration of 1-2×10⁵ sperm/ml (Quinn et al., 1982).

Eggs were microinjected as described previously (Jones et al., 1995) with Ca²⁺-green dextran (10 kDa), or indo-1 dextran (10 kDa) which were obtained from Molecular Probes (Eugene, OR), to final concentrations of 10-30 μM in the egg cytosol. In other systems, pore sizes have been measured using fluorescent markers of varying size (e.g., Wyke et al., 1980; Sauer et al., 1991) and we have used this approach using phycoerythrin (240 kDa) and FITC-albumin (70 kDa) which were obtained from Sigma (St. Louis, MO). All fluorescent probes were dissolved in 120 mM KCl, 20 mM Hepes, pH 7.5 for injection. The fluorescent proteins were washed extensively overnight with several ten-fold dilutions in KCl injection buffer using 30 kDa Centricon cut-off filters and centrifugation at 5000 g (Amicon, Beverly, MA) (to remove lower molecular weight contaminants). Eggs were placed in M2 under oil on the stage of a Nikon Diaphot inverted microscope. Borosilicate glass capillaries (1.5 mm outer diameter × 0.86 mm inner diameter) with an internal filament (Clark Electromedical Instruments, UK) were pulled on a vertical pipette puller (David Kopf Instruments, CA) and the tips broken on cotton wool to give a pipette resistance of 10-50 MΩ (when filled with the injection solution. Micropipettes were inserted into cells by overcompensation of negative capacitance on an electrophysiological amplifier used to monitor membrane potential. A precise, bolus, injection corresponding to 5% of the total cell volume was achieved by using a Pneumatic PicoPump (World Precision Instruments) with success rates of >95%. To allow time for diffusion of dye within the eggs, a period of at least 20 minutes from the time of microinjection was allowed and the eggs were stored in M2 medium at 37°C for not more than 4 hours.

The injected eggs had their zonae removed by a brief (10 seconds) incubation in acid Tyrode’s solution. Eggs were then immediately transferred to a heated chamber (Intracel) at 37°C containing 500 μl of M2 without bovine serum albumin (BSA). The eggs were allowed to attach to the glass base in BSA-free M2 before the addition of 500 μl of M2 containing 14 mg/ml V BSA. In all experiments, complete diffusion of injected dye was confirmed by the uniform distribution of fluorescence throughout the entire egg before adding any sperm to the solution. To record Ca²⁺ changes at fertilization 10 μl of capacitated sperm suspension were added to the chamber and as soon as sperm reached the egg (as observed by conventional light microscopy), fluorescence imaging was started.

A modified Zeiss LSM 410 confocal microscope (Zeiss, Oberkochem) was used which had an Ar-ion and a Ti:Sapphire (Coherent, Santa Clara, CA) laser as light sources. For two-photon imaging, the Ti:Sapphire laser was used with the emission pinhole fully open and excitation wavelength of 690 nm for Indo-1 or 850 nm for Ca²⁺-green. Pulse lengths were less than 100 fs at the sample (see Soeller and Cannell, 1996 for further details). For conventional confocal imaging, the pinhole diameter was set to <1.2 Airey units which provided a similar resolution to that obtained when using 2-photon excitation and a fully open pinhole (approx. 0.35 × 0.35 × 0.7 μm x, y and z respectively). For ratio imaging of indo-1 a 450 nm long pass filter was used to split the emitted light. Ratios were calculated as the ratio of intensity in the long wavelength channel (λ>450 nm) and short wavelength intensity (λ<450 nm). A 40× 1.2 N.A. water immersion objective was used and this objective was heated to 37°C by a warming coil fitted to the objective body. Excitation and emission filters were custom filters manufactured by Chroma (Brattleboro, VT) to the authors’ specifications. Normally, imaging was carried out at pixel dwell times of approx. 2 μseconds and for fast scanning only 10 scan lines were acquired to provide images of a narrow part of the cell at 100 frames/second. The host computer memory for the LSM410 was increased to 32 Mbyte to allow rapid storage of large numbers of images (or in some cases images were written to magnetic disk via cache memory). Image processing and analysis was carried out using custom routines written in IDL (Research Systems, Boulder CO) running on Silicon Graphics Workstations (Mountain View, CA). Long term image archive was provided by 1.2 GByte magneto-optical disks supported by a Silicon Graphics file server.

In all experiments (n>30), eggs were microinjected with either dextran-linked indo-1 or dextran-linked Ca²⁺-green. Following addition of sperm to the heated chamber containing eggs, no detectable sperm autofluorescence was observed. However, after attachment of sperm to an egg, the fluorescence in the attached sperm increased until it became at least as fluorescent as the egg cytoplasm (Fig. 1A,B). The period between sperm addition and attachment to the egg (as observed by conventional light microscopy) was variable, but was not less than two minutes. This delay was due to the time taken for sperm to swim to the egg from their (more distant) site of addition in the chamber. The time interval between sperm attachment and an increase in sperm fluorescence was also variable, with some sperm it was a few tens of seconds while in others it took several minutes. Sperm fluorescence always developed in both the head and the mid piece of the tail, but this observation could not be confirmed at more distal tail pieces because of egg movement (due to other unfused sperm). In all experiments described here, only eggs with a single fused sperm were analyzed, although in a very few cases polyspermy was observed. After a variable delay (1-5 minutes), eggs produced a transient increase in cytoplasmic Ca²⁺ (Fig. 1C). The increase in Ca²⁺ propagated across the cell in about 2 seconds until the entire cytoplasmic Ca²⁺ level was elevated (see below and Fig. 1C). At least a part of the variation in the time taken to produce a Ca²⁺ transient after sperm fusion was probably due to the time taken to find the attached sperm which was complicated by the narrow depth of focus of the microscope.

The time course of propagation of the increase in fluorescence across the egg is examined in Fig. 2. By limiting the region that was examined to just a few lines (Fig. 2A) it was possible to acquire images at 100 frames/second. The time course of fluorescence change in the egg cytoplasm near the sperm and at two more distant sites (indicated in Fig. 2A) are shown in Fig. 2B. At all three sites, fluorescence increased initially exponentially before reaching a peak in about 6 seconds. However, the signal from the site distal to the sperm (green trace) was delayed by about 1-2 seconds relative to the site proximal to the sperm. These data are consistent with the egg cytoplasm behaving as an excitable medium with a wave being initiated near the site of sperm fusion. The propagation velocity of the Ca²⁺ wave across the cytoplasm was approx. 50 μm/second and was consistent with that previously reported for the mouse (47.5±6.06 μm/second; Cheek et al., 1993) but faster than the 16-28 μm/second found in hamster (Miyazaki et al., 1986). Furthermore the fact that the cytoplasm near the sperm responded first would be consistent with the idea that sperm initiates the Ca²⁺ wave from the site of sperm fusion (Miyazaki et al., 1986; Cheek et al., 1993).

If Ca²⁺ influx via the sperm provided ‘trigger’ Ca²⁺ for Ca²⁺ wave initiation, the cytoplasmic Ca²⁺ level in the sperm should increase before the egg or be higher than that in the egg. The first possibility is examined in Fig. 2C which shows the time course of normalized fluorescence change in the sperm and egg cytoplasm near the site of sperm fusion. Although the change in normalized fluorescence from the egg and sperm had a similar amplitude and time course, there was no evidence to support the idea that the sperm Ca²⁺ changes might precede those of the egg. In fact, the sperm signal appeared to lag behind the egg cytoplasm signal by about 0.2 seconds (Fig. 2C). In 3 additional experiments, we could not detect any evidence for a delay between the time course of fluorescence change in the sperm and the egg cytoplasm near the sperm. Although many other studies have shown that Ca²⁺ waves develop after fusion (e.g. Miyazaki et al., 1986), these data are the first demonstration that the sperm also undergoes a Ca²⁺ transient coincident with the initiation of the egg wave. However, the absolute level of fluorescence from the sperm was quite different from that in the egg cytoplasm which would complicate the interpretation of these data (see Fig. 2A). To overcome this problem, dextran-linked indo-1 was used with the fluorescence being recorded at two wavelengths to allow calculation of ratio images (Fig. 3). In Fig. 3A the Ca²⁺ increase in both the sperm and egg can be seen and it is apparent that the amplitude of the change in Ca²⁺ in both the sperm and egg are comparable. Although the signal-to-noise ratio in the indo-1 ratio images was lower and it was not possible to perform a detailed analysis of the kinetics of the Ca²⁺ change, when taken together, the indo-1 and Ca²⁺-green experiments showed that the Ca²⁺ changes in the sperm head had (essentially) the same amplitude and time course as the changes in the egg cytoplasm near the site of sperm fusion. Nevertheless, the absolute indo-1 ratio in the sperm was slightly different from that in the egg, an effect that appeared to be related to the fact that all of the fluorescent indicators (see Figs 4A, 5B,C below also) produced larger signals in the sperm head than in the egg. Even when high signal to noise ratio images were obtained (Fig. 3B) a small difference in Indo-1 ratio remained which did not exclude the possibility that the sperm might be providing Ca²⁺ to the egg.

In cardiac muscle it has been suggested that it is also possible for a Ca²⁺ wave to be initiated by a low amplitude Ca²⁺ influx to induce a local ‘Ca²⁺ overload’ resulting from an increase in cytoplasmic Ca²⁺ being taken up by intracellular stores which then become unstable and initiate wave generation (Fabiato, 1985; Wier and Blatter, 1991; see also Takamatsu and Wier, 1990 and Cheng et al., 1996). In this case, a marked difference between the levels of Ca²⁺ in the sperm and egg (as well as their time course) might not be detected if the permeability of the site of sperm fusion was high (see below). The induction of Ca²⁺ overload in this way should be detectable as a local increase in cytoplasmic Ca²⁺ near the Ca²⁺ source -the sperm. However, the high resolution indo-1 image in Fig. 3B does not support the idea that the egg cytoplasmic Ca²⁺ level near the sperm was higher than at more distant sites. This view was supported by quantification of the ratio levels from the regions marked 1 and 2 in Fig. 3C. The fluorescence ratio in box 1 was 0.54±0.04 and in box 2 was 0.53±0.03 (mean ± s.d., n=1225 pixels). These regions were not therefore significantly different (P<0.01). Similar results were obtained in 3 additional experiments.

Nevertheless, we considered that these data did not rule out the possibility that a very small Ca²⁺ influx from the sperm (‘conduit model’) might remain undetected, and given the problems in accurate interpretation of indo-1 ratio images from different cellular compartments we decided to manipulate extracellular Ca²⁺ to minimize any possible Ca²⁺ influx from the sperm. Eggs placed in a nominally Ca²⁺ free medium also showed a transient increase in intracellular Ca²⁺ after sperm fusion (Fig. 4A). Since the T6 medium used to capacitate sperm (Quinn et al., 1982) contained 1.78 mM Ca²⁺ the dilution of the sperm solution by the solution in the chamber would have increased the extracellular Ca²⁺ level to about 18 μM. Although this low Ca²⁺ should have reduced the possible Ca²⁺ influx into the sperm and egg by more than an order of magnitude it cannot exclude the possibility that some Ca²⁺ influx might still occur. To further reduce Ca²⁺ levels a Ca²⁺ - free bathing medium containing 100 μM EGTA was used. In this solution, the increase in extracellular Ca²⁺ concentration brought about by sperm addition should have been 13 nM (Maxchelator Shareware v4.63). This low level of extracellular Ca²⁺ (see Discussion) should have reversed any gradient of Ca²⁺ from sperm to egg and thereby prevented any calcium influx via the sperm. Although sperm rapidly lost motility in this solution, in 5 out of 8 eggs fusion and a Ca²⁺ transient occurred (as illustrated in Fig. 4B). From these experiments, it follows that an influx of extracellular Ca²⁺ is unlikely to be involved in the initiation of the fertilization Ca²⁺ transient in mouse.

As noted above, the fact that the sperm undergoes a Ca²⁺ transient coincident with the egg transient shows that a significant permeability pathway develops shortly after sperm fusion. Since the fluorescent Ca²⁺ indicators used (≤10 kDa) could readily diffuse into the sperm before the initiation of a Ca²⁺ wave, these data suggest that moderate molecular weight substances can diffuse between the sperm and egg (Lawrence et al., 1997), as required for the ‘content’ model (see Introduction). In conjunction with this point, we verified that diffusion of dye from the egg into the sperm was fast by attempting to selectively bleach the sperm fluorescence. However, attempts to bleach sperm fluorescence by repetitive scanning failed (not shown). This was despite the fact that independent tests showed that fluorochromes could be photobleached when immobilized in gels (Cannell and Soeller, 1997) or in small drops of saline at similar excitation powers. Experiments with even higher molecular weight indicators were performed to estimate the minimum size of the pores associated with this permeability pathway. The first series of experiments used dextran-linked indo-1 as a fusion marker for eggs which also contained FITC labeled albumin (70 kDa). Sperm-egg fusion was detected by monitoring fluorescence of dextran-linked indo-1 in the sperm head by two-photon excitation at 710 nm. In this mode the majority of fluorescence is due to indo-1 (Fig. 5A). When fusion was detected, the excitation wavelength was switched to 488 nm and FITC-albumin fluorescence was measured at 520 nm in the conventional confocal mode of the microscope. The lack of cross-talk between indo-1 and FITC signals was confirmed in eggs that were injected with either indo-1 or FITC-albumin (Fig. 5A). Fig. 5B shows an egg labeled with both indo-1 dextran and FITC-albumin, and it is apparent that both markers readily entered the sperm head. Furthermore, the relative intensity of the FITC-albumin signal between the sperm and egg was as large as the relative sperm-egg indo-1 dextran signal. This suggests that the delay in the passage of FITC-albumin into the sperm must have been comparable to that of the indo-1 dextran and certainly less than the time taken to switch microscope excitation wavelengths (about 5 seconds). We conclude that the pores linking the cytoplasm of the egg and sperm are not only large but also develop rapidly during (or after) sperm-egg fusion.

To further examine the size of the pores linking sperm and egg we used phycoerythrin (240 kDa) whose fluorescence (maximum at 575 nm) is an intrinsic property of the molecule. As shown in Fig. 5C, phycoerythrin also entered the egg cytoplasm at the same time as indo-1 dextran. Therefore, diffusion of these globular protein markers suggest that pores >8 nm in diameter (see Discussion) form rapidly after sperm fusion. Such large pores would allow Ca²⁺ changes in the egg cytoplasm to pass rapidly into the sperm and can explain why the sperm undergoes a synchronous Ca²⁺ transient with the egg (see above). In addition, such pores would permit high molecular weight substances to pass from the sperm to the egg as required for the ‘content’ model.

The present experiments describe observations made on mouse eggs following sperm fusion using a two-photon/confocal imaging system (Soeller and Cannell, 1996). Sperm-egg fusion was observed by the transfer of markers (some of which were used for Ca²⁺ measurement) from the egg into the sperm head. From the time of (measurable) fusion until the first sperm-egg Ca²⁺ transient there was a distinct delay, the latent period, that was at least 1 minute. These observations are in agreement with a recent detailed study of the length of the latent period between sperm fusion and Ca²⁺ changes in the mouse egg (Lawrence et al., 1997). We have excluded both the Ca²⁺ ‘conduit model’ and the ‘Ca²⁺ bomb’ hypothesis for the initiation of the first Ca²⁺ rise by examining local Ca²⁺ changes in the sperm and egg as well as by manipulation of extracellular Ca²⁺ levels. In addition, we found that large diameter pores, which would be obligatory for the ‘content’ model for egg activation, form before any detectable increase in Ca²⁺.

In a small number of eggs (<5%) there was no observable sperm fusion even though the egg was surrounded by many sperm. In such eggs there was never a spontaneous increase in Ca²⁺. Similarly, even in eggs that underwent sperm fusion, there was no spontaneous Ca²⁺ increase in eggs before the fusion event. Therefore, the Ca²⁺ changes that were recorded could not be explained by an artifact associated with two-photon imaging.

The obvious explanation for the increase in sperm fluorescence after sperm-egg fusion is that the fluorescent indicators diffused from the egg cytoplasm into the sperm. In addition, the increase in sperm fluorescence occurred at the emission wavelength of the injected fluorochrome regardless of which fluorochrome was used. This observation makes it highly unlikely that some change in an intrinsic sperm fluorochrome (which would have a unique emission spectrum) was responsible for the development of sperm fluorescence. Our inability to selectively photobleach sperm fluorescence showed that the permeability of the pathway from the sperm to the egg must have been large enough to ensure that diffusion of fresh fluorochrome from the egg (which acts as a large reservoir) prevented significant reduction in fluorochrome levels in the sperm. This view is supported by the ‘pore sizing’ experiments with FITC-albumin and phycoerythrin which suggest that the sperm cytoplasm is readily accessed from the egg cytoplasm by high molecular weight substances.

The two-photon imaging facilitated the use of ratiometric dyes for Ca²⁺ recording by reducing fluorochrome photobleaching. This reduction in photobleaching allowed continuous imaging of the egg while attempting to locate the plane of sperm fusion (as well as during the time which elapsed after sperm addition to the experimental chamber). It was also possible to obtain high quality ratio images (Fig. 3) of the area surrounding the site of sperm fusion and these images showed no detectable local elevation in cytoplasmic Ca²⁺ before the initiation of the Ca²⁺ wave. As noted above, this observation makes it unlikely that the sperm supplies a Ca²⁺ trigger that initiates the Ca²⁺ wave in mouse. Similarly, no measurable Mn²⁺ influx occurs in mouse eggs before the initiation of sperm-induced Ca²⁺ oscillations (McGuiness et al., 1996). Although in sea urchin eggs, addition of La³⁺ or Ca²⁺ chelators block egg activation (Creton and Jaffe, 1995), these apparently conflicting results may be due to non-specific effects of La³⁺ as well as the problematic gradual loss of sperm motility in low Ca²⁺ solutions.

When Ca²⁺ was omitted from the extracellular medium and 100 μM EGTA added, sperm viability was quickly affected so that the majority of sperm were immotile within about 10 minutes. This result is in agreement with detailed studies of sperm viability in various Ca²⁺ buffers (Yanagimachi, 1978; Heffner and Storey, 1981). However in >50% of eggs, sperm fusion still occurred and the first Ca²⁺ wave propagated in the same way as observed in normal Ca²⁺-containing media. These results appear to exclude the possibility that extracellular Ca²⁺ or Ca²⁺ within the sperm head produces the first Ca²⁺ transient in the egg. In addition, it is clear that normal extracellular Ca²⁺ levels are not required for sperm-egg fusion (beyond that being associated with the maintenance of sperm motility). In agreement with this result, in sea urchin, human, guinea pig and mouse, sperm fusion has been achieved in Mg²⁺-containing media with no added Ca²⁺ (Yanagimachi, 1978; Schmidt et al., 1982; Conover and Gwatkin, 1988). It should be noted that M2 medium contains 1.19 mM Mg²⁺ (Quinn et al., 1982), so our results are fully consistent with the lack of an obligatory role of Ca²⁺ in sperm-egg fusion.

It has been argued that the transient elevation of Ca²⁺ associated with sperm addition could preclude exclusion of the‘Ca²⁺bomb’ and ‘Ca²⁺ conduit’ hypotheses (Jaffe, 1990). However, we avoided this complication by adding sperm several millimeters from the egg in an EGTA containing medium. If no mixing occurred, the free Ca²⁺ at the egg would have been even lower than that calculated (13 nM). If the egg and sperm solutions were only partially mixed, the free Ca²⁺ at the site of sperm addition would have been higher than 13 nM but that at the egg would still have been less than 13 nM because bulk stirring did not occur and diffusion is slower than swimming sperm.

In mammals, the protein(s) which are involved in the formation of the fusion pore are not fully characterized (Myles, 1993; Wolfsberg and White, 1996). Candidates include the PH-30 protein which has two subunits, fertilin α and β of 45-49 kDa and 25-33 kDa respectively (Primakoff et al., 1987; Blobel et al., 1992) which are members of the ADAM family (reviewed in Wolfsberg and White, 1996). Other candidates include the DE protein which has two 37kDa subunits (Rochwerger et al., 1992) but several other candidates have been proposed (reviewed by Myles, 1993). Since the pores appear to form without allowing extracellular Ca²⁺ to enter the cell (as no local Ca²⁺ increase near the site of sperm-egg fusion was detected) this raises a question as to how such large pores might assemble from such smaller candidate proteins.

The mechanism of pore formation at gamete fusion may be analogous to other fusion events such as exocytosis (e.g. see Whitaker and Swann, 1993; Betz and Angleson, 1998). It is unlikely that the pores that we have detected are a single molecule since they would be visible with electron microscopy (note that E. coli caseinolytic protease is cylindrical with a pore size of 3.2-3.6 nm and is easily seen in electron micrographs; Flanagan et al., 1995). By analogy with exocytosis, the mouse fusion pore is probably formed by the assembly of protein subunits that are present in both sperm and egg membranes. If this is the case, we suggest that the large quantity of kinetic energy available from sperm motion may power the dislocation of lipid to form a stable pore after protein sub-units in egg and sperm membranes bind. This mechanism would make spontaneous pore fusion extremely unlikely while still allowing motile sperm to achieve fusion. In connection with this point we note that, even when eggs had many sperm attached, no fusion with immotile sperm occurred (not shown).

The sperm-egg pores detected in this study form a path for high molecular weight substances to enter the egg from the sperm. This finding can therefore be considered to be supportive to the idea that the sperm supplies a factor which destabilizes intracellular Ca²⁺ stores to produce Ca²⁺ transients during fertilization (the ‘content’ model). Since the present data excludes the ‘conduit’ model for the initiation of the first Ca²⁺ transient only the ‘content’ model or the ‘contact’ model remain candidates. As noted above, simple ‘contact’ between sperm and egg did not initiate a Ca²⁺ transient unless pore formation also occurred. It follows that a purely ‘contact’ model seems less likely than the ‘content’ model at this time.

We wish to thank the Wellcome Trust for the provision of the confocal/two-photon microscope. We would like to express our thanks to Dr Karl Swann, Department of Anatomy and Developmental Biology, UCL, for critical reading of this manuscript and to Professor David Whittingham, Department of Anatomy and Developmental Biology, SGHMS, for supporting this work.