Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies

Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.     

A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation.

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.     

Role of integrins and polyphosphoinositide metabolism during fertilization in sea urchin egg and hamster oocyte

Summary

In almost all species studied to date, a transient increase in the intracellular free calcium concentration (Ca_i) occurs after fertilization and is essential for egg activation. How the sperm triggers this calcium signal remains, however, to be determined. In this brief review, we compare the mechanisms that are common to mammalian and invertebrate systems. In the light of our own data, we discuss how integrins that have recently been proposed to mediate sperm-egg binding and fusion in mammals might trigger egg activation through cytoskeletal structures. We suggest a model, leading to the calcium signal and common to all species, where phosphorylation on tyrosine and phospholipase Cγ (PLCγ) are interconnected pathways stimulated by a multimolecular complex involving integrins.     

Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei

Eggs of Xenopus laevis were injected with a calcium buffer before insemination, to examine the effect of preventing or suppressing the sperm-induced increase in intracellular calcium on the fertilization potential, exocytosis, and pronuclear formation. Microinjection of BAPTA [(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)] at concentrations between 0.2 and 0.7 mM usually suppressed the fertilization potential to a series of transient depolarizations. The fertilization potential was completely inhibited when the final concentration of BAPTA in the egg was greater than 0.7 mM. These observations support the hypothesis that activation of the chloride conductance responsible for the fertilization potential depends on an increase in intracellular calcium. Exocytosis of cortical granules and elevation of the fertilization envelope were prevented by injecting BAPTA at concentrations greater than 0.2 mM. Injection of BAPTA to suppress the rise in calcium did not inhibit sperm entry and BAPTA-injected eggs were highly polyspermic. Examination by light and electron microscopy revealed that sperm decondensation and pronuclear formation were prevented by injection of the calcium buffer before insemination.     

A calcium‐mediated actin redistribution at egg activation in Drosophila

Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.     

Egg activation in flowering plants

Compared to animals and algae, egg activation in flowering plants is still poorly understood because of the inaccessibility and complexity of the fertilization process which is double and internal. However, the development of in vitro fertilization (IVF) systems in maize and a few other plants, despite some limitations, offers new possibilities for the study of early post- fusional events and signals leading to egg activation under defined conditions. This review reports recent data on calcium events induced by gamete fusion during maize IVF and presents perspectives on the role of calcium in egg activation and in early development. Received: 2 December 2000 / Accepted: 7 June 2001     

Source and sinks for the calcium released during fertilization of single sea urchin eggs

The source and sinks for the intracellular calcium released during fertilization were examined in single eggs from the sea urchin, Arbacia punctulata. Single eggs were microinjected with the calcium photoprotein, aequorin. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or observed with a microscope-image intensifier-video system. In the normal egg a propagated release has been observed. The source of the calcium was investigated in the organelle-stratified centrifuged egg and by the use of mitochondrial uncouplers. In the organelle-stratified centrifuged egg, the calcium-aequorin luminescence was found to originate from the clear zone. The principal constituent of the clear zone is the endoplasmic reticulum. Other potential sources of calcium are the mitochondria. Their contribution to the calcium transient was investigated by exposure of aequorin-injected eggs to mitochondrial uncouplers either before or after fertilization. There was no calcium released from the mitochondria before fertilization. A very large calcium store was released from the mitochondria after fertilization. Interestingly, eggs fertilized in the presence of uncouplers showed no increase in the calcium-aequorin luminescence over untreated eggs. Apparently, in the absence of mitochondrial uptake, other sinks for calcium with affinity and capacity similar to the mitochondria exist, but their nature is unknown. We suggest that the endoplasmic reticulum is the source of the intracellular calcium released upon fertilization and that the mitochondria are the principal sink. The results are discussed with regard to the metabolic activation of the egg.     

Fertilization channels in ascidian eggs are not activated by Ca

Using the whole-cell voltage clamp technique, experiments were carried out on ascidian eggs to determine the role of intracellular Ca in the gating of fertilization channels. Raising the level of Ca by adding Ca to the intracellular perfusion medium or by loading the egg cortex (greater than 50 microM) with Ca through voltage gated channels did not lead to the activation of fertilization channels. Alternatively, eggs exposed to low-Ca seawater, perfused with the chelator K-EGTA or Ca channel blocking agents to prevent the release of Ca from intracellular organelles, and subsequently inseminated generated fertilization currents. This argues against Ca as a second messenger in the activation of fertilization channels in the ascidian egg and alternative mechanisms are discussed.     

Signal transduction at fertilization: the Ca2+ release pathway in echinoderms and other invertebrate deuterostomes

Gamete interaction and fusion triggers a number of events that lead to egg activation and development of a new organism. A key event at fertilization is the rise in intracellular calcium. In deuterostomes, this calcium is released from the egg's endoplasmic reticulum and is necessary for proper activation. This article reviews recent data regarding how gamete interaction triggers the initial calcium release, focusing on the echinoderms (invertebrate deuterostomes) as model systems. In eggs of these animals, Src-type kinases and phospholipase C-gamma are required components of the initial calcium trigger pathway in eggs.     

Teleost fish spermatozoa contain a cytosolic protein factor that induces calcium release in sea urchin egg homogenates and triggers calcium oscillations when injected into mouse oocytes

Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.     

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs.

Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca2+]i). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885-892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca2+ transient. We report here that PKC activators do not induce the intracellular Ca2+ transient, or the activation-associated increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597-604, 1990).     

The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs

Previous work has suggested that the activation of the sea urchin egg at fertilization is the result of a transient increase in intracellular free calcium and an increase in intracellular pH. We have investigated the absence of nuclear activation in incompletely activated eggs and have found a correlation between nuclear activation and the levels of total reduced nicotinamide nucleotides (NAD[P]H). Eggs activated with ammonia show a similar correlation: besides its action as a weak base in raising intracelluar pH (which we conclude is insufficient to stimulate or maintain nuclear activation as judged by nuclear envelope breakdown or DNA synthesis), ammonia increases NAD(P)H. This increase is associated with the stimulation of 6-³H-thymidine incorporation into egg DNA. Removing ammonia decreases NAD(P)H, and tritiated thymidine incorporation ceases. We conclude that a critical level of NAD(P)H is essential to nuclear activation and that the increase of NAD(P)H at fertilization must be included with the increase in calcium and pH as a causal agent in development.     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Fertilization signalling and protein-tyrosine kinases

Fertilization is initiated by species-specific gamete cell recognition, i.e. sperm-egg interaction, followed by a rapid and sustained activation of multiple cellular and biochemical events, collectively called 'egg activation', which is indispensable for successful formation of zygotic nucleus and later embryogenesis. It is well known that sperm-induced egg activation is mediated by a transient release of calcium ions that originates from the sperm entry point and propagates through the entire egg cytoplasm. It is unclear, however, what kind of upstream events prelude to the calcium transient after sperm-egg interaction. Recently, much attention has been paid to the role of protein-tyrosine phosphorylation in egg activation process by a number of studies on some well-established model organisms. These includes marine invertebrates, frogs, and mammals. In this review, we will summarize the recent findings that begin to uncover a 'missing link' between sperm-egg interaction and egg activation with emphasis on the role of egg protein-tyrosine kinases (PTKs) in Xenopus egg fertilization.     

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.     

Egg activation: upstream of the fertilization calcium signal

Interaction of sperm and egg at fertilization induces well-coordinated molecular events including specific recognition between species, adhesion and fusion, that lead to the formation of a zygote, a totipotent cell that develops into a new individual. A calcium signal, common to a great number of species, from marine invertebrates to mammals, is essential to activate the metabolism of the unfertilized oocyte. However, how fertilization triggers this calcium signal and initiates development of the early embryo is far from understood. The signaling pathways activated in eggs may be similar to those described in somatic cells, since changes in intracellular free calcium and in mitosis activating protein (MAP) kinase activity occur in both systems after activation. Several hypotheses are currently proposed, implying a spermatic ligand binding to a specific receptor expressed at the egg surface, or where the fused sperm either allows the transit of external calcium into the egg or injects one (or several) activating factor(s). It is still not known which of these ideas is true. We concentrate in this review on the possible signaling pathways involving IP3 (inositol trisphosphate), since its production is involved in most species to generate the fertilization calcium wave.     

The signalling cascade of fertilization

The early event of fertilization-induced egg activation is a mobilization of intracellular Ca2+ that originates from the sperm entry point and spreads through the entire egg cytoplasm. Recently, this process has been established to require the sequential activation of Src family kinases, phospholipase C gamma, and inositoltrisphosphate receptor of endoplasmic reticulum. This review summarizes recent findings concerning the signalling pathway of fertilization from sperm-egg interaction to the Ca2+ release with emphasis on the role of tyrosine kinases in the egg activation.     

The wave of activation current in the Xenopus egg

A ring-shaped wave of inward current, the activation current, propagates across the Xenopus egg from the site of activation during the positive phase of the activation or fertilization potential. This activation current wave is due to an increased chloride conductance and reflects the propagated of the ionic channels responsible for the fertilization potential. These channels are present in the animal and vegetal hemispheres; however, the magnitude of the activation current is 6-7 times greater in the animal hemisphere. Outward current of a smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of activation where the current is first inward. The inward current wave is detected in all eggs activated by sperm and in eggs activated by pricking with a sharp needle, by application of the Ca2+ ionophore, A23187, and by intracellular iontophoresis of Ca2+ or inositol 1,4,5-trisphosphate. Reduction of the inward current by TMB-8, which blocks intracellular calcium release in some cells, suggests that the activation current channels are calcium sensitive and that the current wave is concomitant with a wave of increased intracellular calcium initiated by sperm-egg interaction. The wave of cortical granule exocytosis and two or more contraction waves follow the current wave.     

Intracellular calcium release at fertilization in the sea urchin egg.

Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5–4.5 μM free calcium 45–60 sec after activation and to last 2$\text{3}\text{?}$ min, assuming equal Ca²⁺ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zerocalcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for observation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 18 μM Ca²⁺. Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.     

The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.