Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca²⁺ channel, in store-operated Ca²⁺ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3 and 5 rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca²⁺ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP₃F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC₅₀ = 0.5 M), indicating that InsP₃F and LPA principally activate store-operated Ca²⁺ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP₃F-, LPA- or thapsigargin-stimulated Ca²⁺ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP₃F-stimulated Ca²⁺ inflow and only a very small enhancement of LPA-stimulated Ca²⁺ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca²⁺ inflow through the previously-characterised Ca²⁺-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.     

Carboxyl-terminal peptide of beta-amyloid precursor protein blocks inositol 1,4,5-trisphosphate-sensitive Ca2+ release in Xenopus laevis oocytes

The effects of Alzheimer's disease-related amyloidogenic peptides on inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) mobilization were examined in Xenopus laevis oocytes. Intracellular Ca(2+) was monitored by electrophysiological measurement of the endogenous Ca(2+)-activated Cl(-) current. Application of a hyperpolarizing pulse released intracellular Ca(2+) in oocytes primed by pre-injection of a non-metabolizable inositol 1,4,5-trisphosphate analogue. The carboxyl terminus of the amyloid precursor protein inhibited inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca(2+) release in a dose-dependent manner. Equimolar beta-amyloid peptides Abeta(1-40) or Abeta(1-42) had no effect, and whereas a truncated carboxyl terminus lacking the Abeta domain was equipotent to the full-length one, a carboxyl terminus fragment lacking the NPTY sequence was less effective than the full-length fragment. The inhibition induced by the carboxyl terminus was not associated with the block of the Ca(2+)-dependent Cl(-) channel itself or compromised Ca(2+) influx. We conclude that the carboxyl terminus of the amyloid precursor protein inhibits inositol 1,4,5-trisphosphate-sensitive Ca(2+) release and could thus disrupt Ca(2+) homeostasis and that the carboxyl terminus is much more effective than the beta-amyloid fragments used. By perturbing the coupling of inositol 1,4,5-trisphosphate and Ca(2+) release, the carboxyl terminus of the amyloid precursor protein can potentially be involved in inducing the neural toxicity characteristic of Alzheimer's disease.     

Evidence for a mitochondrial chromosome in Xenopus laevis oocytes

When Xenopus laevis mitochondria are gently lysed at physiologic ionic strength, mitochondrial DNA (mitDNA) is extracted associated with proteins. Sedimentation and buoyant density studies indicate that proteins are bound to mitDNA at a ratio of about 1/1. This DNA-protein complex visualized by electron microscopy after fixation with glutaraldehyde appears as a relaxed circular molecule consisting of an average of 48 globular particles interconnected by a thin filament.     

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.     

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

Bilirubin may be transported within intracellular membranes of the hepatocyte and may undergo membrane-membrane transfer to gain access to the conjugating enzyme UDP-glucuronyltransferase in the endoplasmic reticulum. We have demonstrated previously that the lipid composition of liposomal membranes incorporating bilirubin substrate influences the rate of transfer and glucuronidation of bilirubin by hepatic microsomes. To examine the mechanism(s) of substrate transfer, we incorporated radiolabelled bilirubin into small unilamellar model membranes of egg phosphatidylcholine or natural phospholipids in the proportions present in native hepatic microsomes. The rate at which bilirubin was transferred to rat liver microsomes and glucuronidated was then examined in the presence of various endogenous compounds that promote membrane fusion. For bilirubin substrate in membranes of egg phosphatidylcholine, the addition of Ca2+ (2 mM) increased the microsomal glucuronidation rate, whereas retinol enhanced microsomal conjugation rates for bilirubin in membranes of both lipid compositions. When the transfer of [3H]bilirubin from dual-labelled liposomes to microsomes was enhanced by Ca2+ or retinol, there was no associated increase in [14C]phospholipid transfer. Thus it appears likely that bilirubin is transferred to the endoplasmic reticulum by rapid cytosolic diffusion or membrane-membrane collisions, rather than by membrane fusion; this process may be modulated by changes in the lipid microenvironment of the substrate or the effective intracellular concentrations of Ca2+ or retinol. The observation that polymyxin B induced concomitant membrane-membrane transfer of [3H]bilirubin and [14C]phospholipid suggests that under certain circumstances membrane fusion or aggregation may promote the movement of lipophilic substrates in hepatocytes.     

Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis

We have used immunocytological techniques to examine the developmental expression of the Ca2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells.     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes.

Ouabain binding was studied in Xenopus laevis oocytes permeabilized by detergents. The behaviour of markers showed that 10 microM-digitonin selectively disrupts the plasma membrane. In the presence of ATP, oocytes permeabilized at 10 microM-digitonin bound no more ouabain molecules than were required to abolish active 86Rb+ uptake in the intact cells. However, the ouabain binding capacity increased approx. 2-fold when inner membranes were disrupted by SDS or excess digitonin, as judged from the accompanying release of the lysosomal marker beta-hexosaminidase. The results suggest that oocytes have a large internal pool of functional sodium pumps.     

Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger.

Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches. I(soc) survived excision into inside-out configuration. If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches. I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho. Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A. These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules.     

非洲爪蟾卵母细胞P2X受体存在的证明

目的：D1中除有P2Y受体介导的成分外,可能还有P2X受体介导的电流参与。本研究的目的在于进一步证实D1电流中是否有P2X受体介导电流的存在。方法：采用双电极电压钳记录方法。结果：Zn^2＋对不依赖于G蛋白之IATP（D1）具有增强作用;重复施加ATP Dl电流逐步减小,而D2电流则逐步增大。在仅有D12之IATP（D2）情况下,重复加ATP后D2电流不但不增大反而有所下降。结论：非洲爪蟾卵母细胞上存在P2X受体。     

The role of Ca2+ in progesterone-induced germinal vesicle breakdown of Xenopus laevis oocytes: the synergic effects of microtubule depolymerization and Ca2+

 By monitoring ⁴⁵Ca²⁺ influx and efflux from oocytes a transient increase followed by a transient decrease in the Ca²⁺-content of progesterone-treated oocytes was observed. Chelation of intracellular Ca²⁺ with EGTA or BAPTA-type buffers inhibited progesterone-induced GVBD. Buffers with a mid-range K_d (∼1.5 μm) were most effective in inhibiting GVBD whereas buffers with a K_d above or below this value were less effective. These observations indicate that intracellular Ca²⁺, probably in the form of a localized release, is required for progesterone-induced oocyte maturation. However, Ca²⁺ alone was insufficient to induce GVBD. When the effects of nocodazole and taxol upon this Ca²⁺-requirement were tested, we observed that taxol-induced microtubule polymerization not only delayed progesterone-induced GVBD but also completely inhibited it in combination with BAPTA-AM. Conversely, nocodazole-induced microtubule depolymerization in combination with ionophore A23187 not only accelerated progesterone-induced GVBD, but also induced GVBD in the absence of progesterone. The combined treatment of oocytes with nocodazole and InsP_3, or with cold treatment and ionophore A23187 also induced GVBD in the absence of progesterone. Thus, Ca²⁺ and microtubule depolymerization synergistically promote GVBD. In both nocodazole- and cold-treated oocytes, the GV was displaced to the periphery of the oocyte and underwent GVBD when treated with A23187. However, when the GV was displaced to the cortex by a centrifugal force under conditions that would not cause microtubule depolymerization and the oocyte was treated with A23187, oocytes did not undergo GVBD. Received: 19 January 1996 / Accepted: 21 May 1996     

Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes.

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300 microns under the membrane), the amplitude of the transient Cl- current induced by the second injection is several-fold higher than that of the first one. This 'potentiation' persists even at 60-90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2(+)-free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.     

Xenopus laevis ovarian CYP17 is a highly potent enzyme expressed exclusively in oocytes. Evidence that oocytes play a critical role in Xenopus ovarian androgen production

Progesterone has long been considered the primary mediator of Xenopus oocyte maturation. We have recently shown, however, that androgens, which are equal or more potent promoters of maturation and are present at higher levels in ovulating frogs, may also be playing an important physiologic role in mediating maturation. Here, we examined the role of CYP17, a key enzyme mediating sex steroid synthesis, in Xenopus ovarian androgen production. We found that the 17,20-lyase activities of Xenopus CYP17 exceeded the 17alpha-hydroxylase activities in both the Delta4 and Delta5 pathways; thus, Xenopus CYP17 rapidly converted pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and androstenedione, respectively. This remarkably robust activity exceeds that of CYP17 from most higher vertebrates, and likely explains why virtually no progesterone is detected in ovulating frogs. Additionally, ovarian CYP17 activity was present exclusively in oocytes, although all other enzymes involved in sex steroid production were expressed almost entirely in surrounding follicular cells. This compartmentalization suggests a "two-cell" model whereby Xenopus ovarian androgen production requires both follicular cells and oocytes themselves. The requirement of oocytes for ovarian androgen production further introduces the unusual paradigm whereby germ cells may be responsible for producing important steroids used to mediate their own maturation.     

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.     

Ca2+-dependence of the depolarization-inducible Na+ current of Xenopus oocytes

The role of Ca²⁺ on the depolarization-induced appearance of a Na⁺ current in Xenopus oocytes was studied. Oocytes were voltage-clamped and the induction of the Na⁺ current was tested under various conditions. In oocytes pre-injected with 400 pmol EGTA to increase the intracellular Ca²⁺ buffering power, the current was significantly reduced. Conversely, when intracellular Ca²⁺ was made to increase by injecting an analogue of inositol 1,4,5-trisphosphate (3-F InsP₃), to cause Ca²⁺ release from internal stores, the induction of the Na⁺ current was potentiated. The depolarization-inducible Na⁺ channels of the Xenopus oocyte membrane appear, therefore, to be Ca²⁺ sensitive, as well as depolarization-activated. J. Cell. Physiol. 174:154–159, 1998. © 1998 Wiley-Liss, Inc.     

STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ Entry

The coupling mechanism between endoplasmic reticulum (ER) Ca(2+) stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1-3]. STIM1 was shown to play a crucial role in this coupling process [4-7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca(2+) entry signals.     

Phospholipase C-gamma1 is required for the activation of store-operated Ca2+ channels in liver cells

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.     

Simulation of the fertilization Ca2+ wave in Xenopus laevis eggs.

In the preceding paper Fontanilla and Nuccitelli (Biophysical Journal 75:2079-2087 (1998)) present detailed measurements of the shape and speed of the fertilization Ca2+ wave in Xenopus laevis eggs. In order to help interpret their results, we develop here a computational technique based on the finite element method that allows us to carry out realistic simulations of the fertilization wave. Our simulations support the hypothesis that the physiological state of the mature egg is bistable, i.e., that its cytoplasm can accommodate two alternative physiological Ca2+ concentrations: a low concentration characteristic of the prefertilization state and a greatly elevated concentration characteristic of the state following the passage of the wave. We explore this hypothesis by assuming that the bistability is due to the release and re-uptake properties of the endoplasmic reticulum (ER) as determined by inositol trisphosphate (IP3) receptor/Ca2+ channels and sarcoendoplasmic reticulum calcium ATPase (SERCA) pumps. When combined with buffered diffusion of Ca2+ in the cytoplasm, our simulations show that inhomogeneities in the Ca2+ release properties near the plasma membrane are required to explain the temporal and spatial dependences of the shape and speed of these waves. Our results are consistent with an elevated IP3 concentration near the plasma membrane in the unfertilized egg that is augmented significantly near the site of fertilization. These gradients are essential in determining the concave shape of the Ca2+ fertilization wave front.