1 Unité de Chronobiologie Théorique, Université Libre de Bruxelles, Faculté des Sciences CP231, Boulevard du Triomphe, Brussels 1050, Belgium
2 Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK
* Author for correspondence (e-mail: gdupont{at}ulb.ac.be)
Accepted 21 April 2004
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Summary |
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Key words: Calcium waves, Fertilization, Ascidian, Sperm factor
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Introduction |
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It is known that in most cell types the signal-induced Ca2+ increases rely on the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], a diffusible second messenger that activates Ca2+ release from the endoplasmic reticulum (ER). As the activity of the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] is also stimulated by cytosolic Ca2+, an initially localized Ca2+ increase triggers the regenerative propagation of the Ca2+ wave throughout the egg while sustained activation of the Ins(1,4,5)P3Rs gives rise to Ca2+ oscillations and waves (Berridge et al., 2000).
A universal trigger for the initial Ca2+ release at fertilization has not yet been identified. Sperm-egg fusion can however be replaced by the injection of a soluble sperm extract (Swann and Parrington 1999; Runft et al., 2002
). In mammals, it has been proposed that the active component of this sperm extract, known as sperm factor (SF), is a new isoform of phospholipase C (PLC) (the enzyme responsible for Ins(1,4,5)P3 synthesis) known as PLC
(Saunders et al., 2002
; Cox et al., 2002
) (reviewed by Kurokawa et al., 2004
). In ascidians, the key factor is an unidentified protein with a size of between 30 and 100 kDa (Kyozuka et al., 1998
). It could be either a soluble PLC or an unknown activator of PLC (Runft and Jaffe, 2000
; Runft et al., 2002
).
Given the pivotal role played by the Ca2+ dynamics in the activation of the egg and its development into an embryo (Dupont, 1998; Ozil, 1998
), a detailed understanding of the biochemical events responsible for the temporal and spatial organization of cytoplasmic Ca2+ signals at fertilization is required. To this aim, simulations provide a useful complementary approach to the numerous experimental studies.
The basic mechanism of Ca2+ oscillations in eggs does not differ much from that of Ca2+ oscillations in somatic cells. Most models ascribe the oscillations to the autocatalytic regulation exerted by cytoplasmic Ca2+ on its own release from the ER (Goldbeter et al., 1990; Sneyd et al., 1995
) (Fig. 1). Some studies, however, stress the possible role of the activation of PLC by Ca2+, a regulation that also leads to a regenerative increase in cytosolic Ca2+ (Meyer and Stryer, 1988
; Hirose et al., 1999
). Despite numerous theoretical approaches (reviewed by Schuster et al., 2002
), no model until now has focussed on the rather typical shape of the repetitive Ca2+ waves that are triggered at fertilization.
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Theoretically, Ca2+ wave propagation can be ascribed to the same regulatory processes as oscillations (Sneyd et al., 1995; Goldbeter, 1996
). It is clear, however, that the detailed characteristics of the waves much depend on the cell type. In eggs, these waves can take the form of sharp fronts, spirals or tides (Lechleiter et al., 1991
; McDougall and Sardet, 1995
; Fontanilla and Nuccitelli, 1998
). The detailed shape of the front can also vary: both convex and concave fronts have been observed (Stricker, 1999
). Moreover, the large size of the eggs allows a clearer manifestation of the effects related with the spatial inhomogeneity of the cytoplasm. Distinct subcellular regions that repetitively initiate Ca2+ waves have been identified in the ascidian egg (McDougall and Sardet, 1995
; Dumollard and Sardet, 2001
; Dumollard et al., 2002
). Three such regions, called `calcium wave pacemakers (PM)', have been reported. (1) PM1 is defined as the initiation sites of the first series of Ca2+ oscillations (series I). PM1 is a moving Ca2+ wave pacemaker: the fertilization wave indeed initiates at the site of sperm entry, while the initiation sites of the subsequent waves progressively migrate with the sperm aster towards the vegetal contraction pole. (2) Pacemaker PM2 is stably localized in the vegetal contraction pole, a cortical constriction of 15-20 µm in diameter. It is a region of dense ER and mitochondria accumulation. (3) An artificial pacemaker called PM3 is located in the animal hemisphere, and is defined as the cellular region most sensitive to an artificial stimulation by Ins(1,4,5)P3. It probably corresponds to a region rich in ER clusters, present around the meiotic spindle in the mature unfertilized egg (Dumollard and Sardet, 2001
).
Some theoretical models have already investigated the spatial characteristics of the Ca2+ increase occuring at fertilization (Wagner et al., 1998; Bugrim et al., 2003
; Hunding and Ipsen, 2003
). It was shown that the correct shape of the fertilization wave in Xenopus oocyte can be reproduced by assuming that Ins(1,4,5)P3 is locally generated at the fertilization site (Bugrim et al., 2003
). Moreover, these studies emphasize the role of the spatial inhomogeneities in the ER distribution (Bugrim et al., 2003
; Hunding and Ipsen, 2003
), in the Ins(1,4,5)P3Rs distribution (Bugrim et al., 2003
) or in Ins(1,4,5)P3 production (Wagner et al., 1998
) to reproduce the experimentally observed spatial profiles. None of these studies, however, deals with repetitive Ca2+ waves, as those observed at fertilization of many species, including ascidians and mammals. In the case of the Xenopus oocytes, the fertilization Ca2+ wave is indeed seen as a switch in a bistable system. Recovery, i.e. return to the basal Ca2+ level, is seen to occur on a much longer time scale than the increase in Ca2+.
In the present study, we simulate repetitive Ca2+ waves with a spatio-temporal pattern analogous to that of series I oscillations in ascidian eggs. We focus on the Ca2+ waves induced by Ins(1,4,5)P3 or its poorly metabolizable analogue glycero-myo-phosphatidylinositol 4,5-bisphosphate [gPtdIns(4,5)P2] and on series I Ca2+ oscillations. We first simulate an existing model for Ca2+ and Ins(1,4,5)P3 dynamics and show that it can reproduce the experimentally observed Ca2+ waves triggered by flash photolysis of Ins(1,4,5)P3 or gPtdIns(4,5)P2 when considering an appropriate inhomogeneous distribution of ER. We then show the results of simulations of the model predicting the effect of a localized injection of a large amount of gPtdIns(4,5)P2. This prediction is confirmed experimentally. In a second part, we use our model to simulate the fertilization wave and the series I Ca2+ oscillations. We find that the best agreement with the experimental data is obtained if it is assumed that the SF in ascidian eggs is a Ca2+-sensitive, highly diffusible PLC, and that PtdIns(4,5)P2, the substrate for PLC, is homogeneously distributed in the whole egg.
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Model |
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![]() | (1) |
where k+ is the rate of inhibition of the Ins(1,4,5)P3R by cytosolic Ca2+ and k- the rate of relief from this inhibition. Activation of the receptor by Ca2+ is assumed to be instantaneous and characterized by a threshold constant Kact. The level of cytosolic Ca2+ (Ccyto) varies through Ca2+ release via the Ins(1,4,5)P3Rs and Ca2+ pumping by Ca2+ ATPases located in the membrane of the ER. We do not consider Ca2+ exchanges with the extracellular medium, as it is known that Ca2+ oscillations in ascidian eggs can occur in the absence of extracellular Ca2+ (Speksnijder et al., 1989; Carroll et al., 2003
). Taking diffusion into account, the evolution equation for the concentration of cytosolic Ca2+ can be written:
![]() | (2) |
In this equation, IRa represents the fraction of Ins(1,4,5)P3Rs in an active state, and is given by:
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where
![]() | (3) |
where IP and gPtdIns(4,5)P2 represent the Ins(1,4,5)P3 and the gPtdIns(4,5)P2 concentrations, respectively. gPtdIns(4,5)P2 is explicitly considered in the model, because we simulate experiments of flash-photolysis of this poorly metabolizable analogue of Ins(1,4,5)P3. Parameter k1 fixes the rate of Ca2+ release through the Ins(1,4,5)P3R and k1b is a leak term; Clum stands for the concentration of free Ca2+ in the ER. The second term of equation (2) represents the pumping of Ca2+ into the ER by the ATPases. The last term is a classical Fick term for diffusion.
The distribution of ER is taken into account through two parameters: and
, which are both functions of space (r) to allow heterogeneity. Two parameters are necessary because the amount of ER affects both the number of Ins(1,4,5)P3Rs and Ca2+ pumps (located in the ER membrane), and the local volumes of the ER and cytosol, respectively. Thus, the first parameter (
) scales the number of channels [Ins(1,4,5)P3Rs and Ca2+ pumps] and similarly affects all flux terms across the ER membrane. The other parameter (
) is defined as the local ratio between the volumes of the endoplasmic reticulum and the cytosol. It accounts for example for the fact that the release of a given number of moles of Ca2+ from the ER will induce a lower increase of the Ca2+ concentration in the cytosol than the associated decrease of Ca2+ concentration in the ER, due to the different volumes of these compartments. In this study, we always assume that the distribution of ER follows the same distribution than that of Ca2+ channels (Fink et al., 2000
). Thus, there is a fixed relationship between
(r) and
(r): assuming a spherical shape for the ER, any change in `
' of a factor `x' will be accompanied by a change in
of a factor x2/3, given the surface/volume ratio of a sphere. In the following, we will discuss changes in ER distribution in terms of
.
In contrast to other models, we do not assume that the Ca2+ concentration inside the ER remains constant, as the fertilization Ca2+ wave implies a massive release of Ca2+ from the ER. In our model, the evolution of the concentration of Ca2+ inside the ER lumen (Clum) is given by:
![]() | (4) |
Implicit in the latter equation is the fact that the ER behaves as a continuous compartment invading the whole egg. In Equations (2) and (4), all fluxes must be seen as effective ones, as Ca2+ buffers are not explicitly incorporated in the model.
Finally, Ins(1,4,5)P3 is assumed to be synthesized from PtdIns(4,5)P2 by PLC and degraded by both a phosphatase and a kinase. Thus, the evolution of the concentration of Ins(1,4,5)P3 (IP) follows:
![]() | (5) |
IIP [for Ins(1,4,5)P3 input] allows us to simulate the experiments of Ins(1,4,5)P3 flash-photolysis (in this case, this parameter takes a non-zero value during the simulated flash). VPLC represents the basal rate of Ins(1,4,5)P3 synthesis in a non-fertilized egg. In equation (5), this rate is assumed to be Ca2+-insensitive, in the absence of further indication as to the PLC isoform present in the ascidian egg. However, given that VPLC is a small term [because it must lead to a basal low level of Ins(1,4,5)P3] the behaviour of the model remains unchanged if VPLC is made Ca2+-sensitive. V5P and V3K stand for the maximal rates of Ins(1,4,5)P3-5-phosphatase and -3-kinase, respectively, while K5P and K3K are the Michaelis constants of the same enzymes. Stimulation of Ins(1,4,5)P3-3-kinase activity by Ca2+ is taken into account; the constant for half-maximal activation is represented by KA3K. As discussed below, the concentration of PtdIns(4,5)P2 (the substrate for PLC) is assumed to be homogeneous throughout the whole egg and to remain constant or at least not limiting (Xu et al., 2003).
Inhomogeneities in the endoplasmic reticulum distribution
The ascidian egg is a large cell, the diameter of which is comprised between 100 and 150 µm. As many other eggs, the cytoplasm is highly structured in specific domains that host different concentrations of intracellular organelles (mitochondria, yolk platelets and endoplasmic reticulum). It is also known that a spectacular reorganization of these egg structures occurs at fertilization (Roegiers et al., 1999). In particular, a wave of cortical contraction leads to the formation of the contraction pole in the vegetal hemisphere, a region containing an accumulation of cortical ER (Roegiers et al., 1999
; Dumollard and Sardet, 2001
). As we focus here on the Ca2+ changes induced by the injection of Ins(1,4,5)P3 or Ins(1,4,5)P3 analogues, or occurring just after fertilization, we do not consider these rearrangements in the present study. As a first hypothesis, we only consider inhomogeneities in the ER distribution (parameters
and
) to test if those are sufficient to account for the observed Ca2+ wave initiation sites. To simplify the simulations, we have chosen a 2D geometry. This assumption amounts to looking at a slice through the egg, but introduces a bias on the value of the fluxes. However, as most parameter values are not known for the ascidian eggs, we think that this assumption (which much reduces the computing time) is worthwhile.
The ER is modelled as a continuous network of varying density throughout the cytosol. Thus parameter , defined as the ratio between the volumes occupied by the endoplasmic reticulum and by the cytosol, is a function of space (r). As suggested by direct observations on mature eggs (Dumollard and Sardet., 2001
; Sardet et al., 2002
), we assume that the density of the reticulum is higher in the cortex than in the cytosol. The thickness of this higher density region is of a few microns. In the model, it is assumed that there is a gradient of reticular density from the periphery to the centre, given by:
![]() | (6) |
where B is the basal density of ER,
C reflects the amplitude of the gradient, rc is the radius of the egg and w reflects the steepness of the gradient. The spatial coordinate r is calculated in a system centred on the centre of the egg.
Moreover, we consider another type of inhomogeneity strongly suggested by recent experimental observations; it has been shown that the artificial pacemaker located in a broad cortical region of the animal hemisphere (PM3) serves as the initiation site of the Ca2+ waves induced by exogenous injections of Ins(1,4,5)P3 or gPtdIns(4,5)P2 (Dumollard and Sardet, 2001). We thus use numerical simulations to investigate the hypothesis that a higher density of ER suffices to explain all the observations performed with respect to this new pacemaker. To account for the possible existence of such a region, we assume that
is maximal at the cortex of the animal pole and linearly decreases towards the centre of the egg (vertical variation only). Thus, in the region called pacemaker 3 (PM3),
![]() | (7) |
The resulting distribution of the ER for our particular set of parameter values is shown in Fig. 2. The average density of the ER is 8%, which is of the order of experimental measurements performed in other cell types (Depierre and Dallner, 1975; Fink et al., 2000
). The resting cytosolic and luminal [Ca2+] result from an equilibrium between Ca2+ fluxes across the ER membranes; in consequence, the steady-state level of Ca2+ in the absence of stimulation depends on ER density and this level of Ca2+ is inhomogeneous both in the cytosol and in the lumen. Although in the ER this inhomogeneity is not significant, cytosolic Ca2+ varies from 0.106 µM to 0.167 µM depending on the ER density (the higher the density, the higher the concentration). This effect is due to the locally lower cytosolic volume in the regions of high ER density (parameter
). By contrast, variations in
affect both release and pumping and do thus not affect the steady-state levels of Ca2+.
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Sperm factor
In the last part of our study, we investigate the sperm factor hypothesis. We start from the basic assumption that SF is a soluble PLC (Swann, 1996; Saunders et al., 2002
; Cox et al., 2002
; Howell et al., 2003
). Thus, we simulate fertilization by assuming that a large amount of PLC is locally introduced in the egg. This additional PLC is superimposed on the basal PLC activity of the egg. SF is assumed to diffuse and to be degraded. The value of the diffusion coefficient will be discussed later. The time scale of SF degradation is chosen to fit the observed duration of series I Ca2+ oscillations in ascidian eggs. We also made the assumption that the activity of the injected PLC is stimulated by cytosolic Ca2+ (see below for the justification of this hypothesis). Thus, when modelling fertilization, a new equation describing the evolution of the level of sperm factor activity is introduced:
![]() | (8) |
where kSF stands for the rate constant of SF degradation and DSF for the diffusion coefficient. Introduction of SF occurs through appropriate initial conditions. Moreover, equation (5) is modified to take into account the PLC activity of the SF:
![]() | (5') |
KASF is the threshold constant characterizing the stimulation of the PLC activity of the SF by Ca2+. The values of VPLC and VSF are taken as constant in the whole egg, based on the assumption that PtdIns(4,5)P2 is homogeneously distributed (see `Simulation of the series I Ca2+ oscillations induced by fertilization').
Simulation method
Numerical simulations have been performed using a variable time-step Gear method. To simulate diffusion, the Laplacian is made discrete using the finite difference method. The egg is divided into mesh points, using a cartesian grid with no flux boundary conditions. The circular shape of the egg is reproduced by applying the appropriate no-flux boundary conditions at all grid points located at a given distance from the centre (corresponding to the egg radius). The egg radius is 75 µm and the mesh size is 1.5 µm.
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Results |
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Also in agreement with a relatively fast degradation of Ins(1,4,5)P3, activation of the eggs by a single injection of Ins(1,4,5)P3 cannot induce repetitive Ca2+ waves, neither in the model, nor in the experiments. As modelled above (Fig. 3) and observed in the experiments (Dumollard and Sardet, 2001), when the Ins(1,4,5)P3 flashes are localized, the Ca2+ wave originates from the site of Ins(1,4,5)P3 release. More surprisingly, when Ins(1,4,5)P3 or its poorly metabolizable analogue gPtdIns(4,5)P2, is homogeneously increased in the whole egg, the Ca2+ wave is always seen to originate from the cortex, in a broad area near the animal pole of the eggs (Dumollard and Sardet, 2001
). As proposed in the experimental study, the existence of this pacemaker revealed by an artificial type of stimulation (called PM3) can be ascribed to a denser distribution of ER near the cortex of the animal pole. We have tested this hypothesis in the model. In Fig. 4, gPtdIns(4,5)P2 is assumed to be homogeneously released in the whole egg. However, the Ca2+ wave clearly initiates in the region with the highest density of ER (see Fig. 2 for the distribution of the ER). The shape of this region of higher density (see `Inhomogeneities in the endoplasmic reticulum distribution') has been fitted in the simulations to get the best agreement with the experimentally observed forms of the Ca2+ wave. The best results are thus obtained when the gradient in
(ER density) is only vertical (along the animal-vegetal axis). Equally important for the appropriate shape of the Ca2+ wave is the slightly more elevated quantity of ER in the cortex. Because of this inhomogeneity, the wave propagates faster in the periphery, allowing for the transformation of a convex front at the onset of the propagation into a slightly concave one as the wave spreads through the egg (Fig. 4), as seen in the experiments.
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From a theoretical point of view, (ER density) is a bifurcation parameter. In other words, increasing the value of
qualitatively changes the behaviour of the system, from resting, to excitable and finally to oscillatory. Thus, at a given fixed level of stimulation [Ins(1,4,5)P3 or gPtdIns(4,5)P2], the cytoplasm is excitable for eliciting a Ca2+ wave only when
is above a critical level. The values of
and of the stimulation intensity used in Fig. 4 are such that PM3 is the only part of the egg initially able to generate a Ca2+ spike. In the rest of the cytoplasm and in the cortex, the synergy between this low level of stimulus and the Ca2+ increase coming through diffusion from an adjacent region of the cell is required to generate a Ca2+ spike.
The level of stimulation [simulated here in the form of the amplitude of the gPtdIns(4,5)P2 influx] can also be viewed as a bifurcation parameter. The model accounts for the experimental observation (Dumollard and Sardet, 2001) that the global amplitude and the propagation velocity of the Ca2+ wave both increase with the amount of gPtdIns(4,5)P2 released into the egg (Fig. 5). These increases in Ca2+ wave amplitude are associated with a widening of the Ca2+ front but the local maximal amplitudes do not change when varying the level of stimulation (not shown). The wave-like behaviour exemplified in Fig. 4 is restricted to a limited range of stimulation levels. In both the model and the experiments, if the magnitude of the global gPtdIns(4,5)P2 increase is too low (below 0.03 µM second-1), it only generates a spatially limited Ca2+ increase confined to the animal pole region. By contrast, for the largest influx terms (above 0.05 µM second-1), the Ca2+ increase occurs simultaneously in the entire egg. For all situations represented in Fig. 5, the local variations of ER Ca2+ associated with the wave of cytosolic Ca2+ are very small given the high level of Ca2+ in this compartment. We have taken an initial value of 875 µM for the Ca2+ concentration in the ER (Montero et al., 1995
; Hofer and Schulz, 1996
).
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In contrast to Ins(1,4,5)P3, gPtdIns(4,5)P2 is very slowly metabolized and its level remains elevated for a long time. As a consequence, several Ca2+ spikes can be generated by a single flash. The model can reproduce this behaviour if the rate of degradation of gPtdIns(4,5)P2 is assumed to be ten times smaller than that of Ins(1,4,5)P3 degradation as it was found experimentally (Bird et al., 1992). Fig. 6A indeed shows that in response to a localized gPtdIns(4,5)P2 increase of high amplitude, two Ca2+ waves are generated; the first one originates from the region of stimulation (here chosen to be in the vegetal pole), while the second one starts in the region of higher ER density in the animal pole region (PM3). A close look at the evolution of the variables of the model indicates that at that time (125 seconds), gPtdIns(4,5)P2 is homogeneously distributed in the egg; the second wave thus originates from the region that is the most sensitive to a homogeneous level of gPtdIns(4,5)P2. This prediction is corroborated by the experimental results shown in Fig. 6B.
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Injections of large amounts of gPtdIns(4,5)P2 mimic the temporal pattern of Ca2+ seen at fertilization
Large amounts of gPtdIns(4,5)P2 globally released in the egg induce a complex series of Ca2+ increases that strikingly resemble the first phase of Ca2+ waves observed at fertilization of ascidian eggs (Dumollard and Sardet, 2001). Fig. 7 shows the simulation of the injection of a large amount of gPtdIns(4,5)P2 (red trace). At time 0, the concentration of the latter compound is increased up to a high value; it then decreases to zero according to equation (5), in which the maximal velocities have been adapted for gPtdIns(4,5)P2 [instead of Ins(1,4,5)P3; see legend to Fig. 4]. The high level of gPtdIns(4,5)P2 causes a long-lasting Ca2+ increase, followed by two shorter spikes (black trace). Also visible are the changes of Ca2+ concentration within the ER (blue trace); note that Ca2+ depletion of the ER (up to
40%) is significant only for the first Ca2+ increase. As far as the spatial aspects are concerned, the first massive Ca2+ increase propagates so rapidly that it seems to occur quasi-instantaneously in the whole egg. The second (and other subsequent) peaks clearly originate from the cortical area of the animal hemisphere, which possesses the highest ER density. These theoretical results are in full agreement with the experimental results (see Fig. 3B of Dumollard and Sardet, 2001
).
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Simulation of the series I Ca2+ oscillations induced by fertilization
Given the observed agreement between our simulations and the experimental observations, we have used the model to make some theoretical predictions about the possible nature of the sperm factor (SF). We simulate fertilization as a localized rise in SF concentration from zero up to an arbitrary value. If we assume that the SF is a Ca2+-sensitive PLC that can diffuse in the cytosol [equations (5') and (8)], the model reproduces the temporal pattern of cytosolic Ca2+ changes observed at fertilization of ascidian eggs (Fig. 8). The level of PLC activity rises instantaneously (corresponding to the injection of SF) and then decays exponentially, due to the first-order degradation term. As for Ins(1,4,5)P3, it massively rises with the step-wise increase in PLC; it then globally decays due to its catabolism by the Ins(1,4,5)P3 3-kinase and 5-phosphatase and to the decrease in PLC. However, because of the stimulation of PLC activity by Ca2+, the concentration of Ins(1,4,5)P3 oscillates in synchrony with Ca2+ oscillations (Meyer and Stryer, 1988).
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The spatial properties of the Ca2+ oscillations shown in Fig. 8 have not yet been investigated in the model. Simulations of the moving pacemaker observed in series I Ca2+ oscillations (PM1) of the ascidian egg indeed require both additional hypotheses and new simulation techniques. It may be that the source of Ins(1,4,5)P3 is moving along the cortex as a consequence of the cortical contraction-induced movement of the sperm aster towards the vegetal hemisphere (Dumollard and Sardet, 2001; Dumollard et al., 2002
; Carroll et al., 2003
).
We have assumed that the Ca2+-sensitive PLC supposed to represent the SF has a relatively high diffusion coefficient (150 µm2 second-1). If this is not the case, partial Ca2+ waves propagating only in the region of the egg opposite to the site of SF increase are observed, while the [Ca2+]c remains constantly elevated in the region closer to the injection site (not shown). This is owing to the fact that far away from the site of SF increase, the level of Ins(1,4,5)P3 is still in the oscillatory regime, because the level of PLC in this region is relatively low as significant diffusion has not yet occurred.
Finally, we have investigated in the model the effect of changing the amount of SF injected into the egg. As shown in Fig. 9, our preliminary model predicts that the amount of SF influences the shape of the first large fertilization spike. As expected intuitively, the duration of the fertilization spike increases with the dose of SF introduced into the egg. Interestingly, the shape of the spike also depends on the dose of SF. The number of small-amplitude spikes superimposed on the plateau increase of Ca2+ rises if the dose of SF decreases. For lower doses of SF (Fig. 9A), the level of Ins(1,4,5)P3 is close to that able to induce oscillations. By contrast, if [SF] is large (Fig. 9B), the Ins(1,4,5)P3 concentration is so high that Ca2+ remains at a high steady-state level set by the actual Ins(1,4,5)P3 concentration.
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Discussion |
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Besides the experimental evidences in favour of the fact that the SF would be a PLC-like factor or its activator, we have ruled out the possibility that the SF in ascidians could be a molecule similar to Ins(1,4,5)P3. We have indeed seen above [see section `An artificial pacemaker site (PM3) is revealed by flash-photolysis of Ins(1,4,5)P3 or gPtdIns(4,5)P2'] that Ins(1,4,5)P3 appears to be a local messenger that cannot, through a single increase, induce repetitive Ca2+ rises. By contrast, a theoretical study of the fertilization Ca2+ wave in
Xenopus oocyte (Bugrim et al., 2003) concludes that an elevated concentration of Ins(1,4,5)P3 near the site of fertilization appears as the most probable mechanism to reproduce the experimental observations. The contradiction between the latter theoretical results and ours can be explained by the fact that the study for Xenopus oocytes simulates the unique fertilization wave as a switch between a stable state with low cytosolic Ca2+ and another stable state with a high cytosolic Ca2+. The passage from one state to the other can, in this case, be induced by a sufficient perturbation, i.e. the initial localized rise in Ins(1,4,5)P3. This hypothesis cannot hold for fertilization in ascidians or in mammals, where wave propagation must obviously be associated with an oscillatory (and not a bistable) dynamic.
The main conclusions that can be drawn from this second part of the study are summarized as follows.
In conclusion, our study demonstrates that when Ins(1,4,5)P3 is produced throughout the whole ascidian egg, spatial inhomogeneities in the ER distribution are responsible for the appearance of the artificial Ca2+ wave pacemaker PM3 in the animal pole of the egg and dictates the spatio-temporal characteristics of the Ca2+ waves triggered by this pacemaker. This model also predicts that the activity of the natural pacemaker PM1 induced by fertilization is regulated by a soluble Ca2+-activated PLC that is injected into the egg. This PLC should hydrolyse PtdIns(4,5)P2 in the whole egg and its activity would oscillate leading to oscillatory changes in Ins(1,4,5)P3 mediating PM1 function. This latter prediction can only be tested by monitoring the spatio-temporal variations of Ins(1,4,5)P3 levels in a single egg undergoing fertilization. In the future, the model can be extended to investigate the origins of the detailed characteristics of the Ca2+ waves in the eggs of different species, for example, the periods of the waves, the existence of pacemaker zones other than those obseved in ascidian eggs or the shapes of the Ca2+ spikes.
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Acknowledgments |
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References |
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