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Egg Activation

Douglas Kline, Kent State University, Kent, Ohio, USA


The interaction between sperm and egg at fertilization stimulates the egg to begin development, a process known as egg activation. The fertilizing sperm not only contributes its complement of chromatin at fertilization, but also provides the signal that releases the egg from meiotic arrest and permits embryonic development.

Secondary article
Article Contents
. Introduction . Importance of Calcium Ions in Egg Activation . Inositol Trisphosphate . Release of Bound Calcium from Egg Storage . Calcium Waves . Proposed Mechanisms for Initiation of Egg Activation: PLCc and Tyrosine Kinase . Proposed Mechanisms for Initiation of Egg Activation: Sperm-borne Activating Factors . Summary

Introduction
Prior to fertilization, the egg is relatively quiescent and is arrested at a specic stage of the meiotic cell division cycle. Developmental biologists have long been interested in determining how the activation signal is conveyed from the sperm to the egg. The process of egg activation has been examined in many species of animals and in a number of plant species. The universal signal for egg activation is an increase in calcium ions within the egg cell. Calcium is primarily released from intracellular stores, although an inux of calcium from the surrounding medium may be important in some cases. In most species, one or many waves of elevated calcium pass through the egg. While there is overwhelming evidence for the central role of calcium in egg activation, the rest of the signalling pathway has not been completely established. Research is now aimed to identify the signalling steps prior to the elevation of intracellular calcium. For many species, there is strong evidence that calcium is released from a pool of calcium in the endoplasmic reticulum (ER) following the binding of inositol 1,4,5-trisphosphate (IP3) to receptors in the ER membrane. The enzyme phospholipase C (PLC) produces IP3 from the membrane lipid phosphatidylinositol bisphosphate, indicating a role for PLC and IP3 in the signalling pathway. Recent research has focused on dening a signalling pathway that could activate PLC in the egg. Other studies have concentrated on determining what factor or factors from the sperm might cause the activation of PLC, induce IP3 production, or initiate calcium release in some other way.

of egg activation is the calcium-stimulated fusion of cortical granules with the egg plasma membrane. The granules release their contents, which contain enzymes that modify extracellular coats around the egg cell to prevent the entry of supernumerary sperm (a mechanical block to polyspermy). Some species utilize both fast and slow blocks to polyspermy, while others, including mammals, rely only on a slow block to polyspermy. Other calciumdependent activation events are so-called later activation eects and these include the recruitment and translation of stored maternal messenger ribonucleic acids (mRNAs), release from meiotic arrest, and the transition to the mitotic cell cycle. The importance of calcium was suggested by observations of pioneering embryologists working in the late nineteenth and early twentieth centuries. These early investigators found that articial activation of unfertilized eggs could be achieved by increasing calcium inux or changing the pH. Many years later, the central role of calcium in egg activation during normal fertilization was established in several species by demonstrating that: (1) there is a natural rise in intracellular calcium in the egg at fertilization; (2) egg activation can be prevented by inhibiting the increase in intracellular calcium; and (3) articially increasing calcium in the egg initiates activation in the absence of sperm.

Importance of Calcium Ions in Egg Activation


The increase in intracellular calcium is responsible for initiating the events of egg activation. In some species, one of the early consequences of the calcium rise is the opening of calcium-gated ion channels in the membrane. The subsequent change in membrane potential (the fertilization potential) may serve to prevent additional sperm from fusing with the plasma membrane (the fast or electrical block to polyspermy). In many species, another early event

Intracellular calcium increases at fertilization


Although the importance of calcium was suggested much earlier, measurement of the natural increase in intracellular calcium at fertilization did not come until much later. Recordings of increased intracellular free calcium were made in the egg of the freshwater sh, medaka, with the photoprotein aequorin in the late 1970s. Measurements have now been made with aequorin or uorescent probes for calcium in many species ranging from starsh to frog, humans and owering plants.
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Egg Activation

Inhibition of the calcium increase prevents egg activation


Egg activation can be prevented by introducing a calcium chelator into the egg before fertilization. Preventing the increase in free calcium, by chelating it immediately on release, prevents the exocytosis of cortical granules and blocks resumption of the cell cycle. Injection of antibodies that block the function of the IP3 receptor also prevents egg activation, demonstrating the importance of both calcium and IP3 for activation of the egg.

Artificially increasing calcium triggers egg activation


Articially increasing calcium within the unfertilized egg initiates activation of the egg. Early experiments used the calcium ionophore A23187 to elevate calcium within the egg. Injection of calcium or IP3 is also eective in initiating activation. In some cases, it has been possible to achieve parthenogenetic development by articially increasing intracellular calcium. For example, classic experiments demonstrated that the frog egg could be activated and undergo parthenogenetic development to the tadpole stage if pricked with a bloody needle in a calcium-containing medium. The elevation of calcium triggered resumption of the cell cycle and the bloody needle contained microtubule organizing centres from disrupted blood cells, which allowed mitosis. Articially elevating calcium in the eggs of some species, particularly mammals, is not always eective in activating the egg. Only the early activation events are triggered in the mouse egg by calcium or IP3 injection. In these cases, cell cycle resumption is incomplete, suggesting that the activation sequence cannot be entirely mimicked by injecting calcium or IP3. However, it has been shown that one of the most eective activating agents is adenophostin, a synthetic analogue of IP3, which does cause the full resumption of meiosis, possibly because it causes prolonged calcium oscillations, which are characteristic of mammalian egg activation (Sato et al., 1998).

the sea urchin egg. Other experiments later demonstrated that PIP2 production also occurs in the frog egg. Microinjection of IP3 can initiate activation of the sea urchin egg and causes calcium release in a variety of species. The natural increase in IP3 concentration at fertilization is more dicult to demonstrate, but recent experiments in frog eggs have conrmed that IP3 is produced at fertilization. Complete inhibition of calcium release in hamster eggs can be achieved by injection of an antibody against the IP3 receptor. Heparin and pentosan polysulfate (although less specic inhibitors of the IP3 pathway) block calcium release in frog and sea urchin eggs, further suggesting that calcium release is mediated by IP3. Inhibiting the enzyme PLC during fertilization of sea urchin and mouse eggs prevents the sperm-induced increase in intracellular calcium, providing further evidence for the central role of IP3. Together with the evidence that a function-block antibody to the IP3 receptor prevents calcium release and egg activation in mammalian eggs, the role for inositol phospholipid turnover and IP3 production is quite strong. IP3 binds with high anity to one of three IP3 receptor subtypes which are the calcium channels that traverse the membrane of calcium storage compartments formed from the ER. The IP3-gated calcium channel exists as a complex of four IP3 receptor subunits, which may be homomeric or heteromeric in somatic cells. Both egg activation and calcium release are completely prevented by the functionblocking monoclonal antibody 18A10 against the type 1 IP3 receptor. Analysis of IP3 receptor subtypes has only been done in several mammalian species. Western analysis has shown that the type 1 IP3 receptor isoform is the predominant IP3 receptor protein expressed in the mouse egg. Some evidence indicates that the type 2 and type 3 receptor might be present, but it is not known if these subtypes are important in calcium release in eggs as either homomeric channels or, together with the type 1 receptor, as heteromeric channels.

Release of Bound Calcium from Egg Storage


The storage compartment for calcium in the egg is the ER. It has been shown that isolated ER from the sea urchin egg eectively releases and sequesters calcium (Terasaki and Sardet, 1991). The ER is known to have calcium pumps, luminal calcium-binding proteins, and calcium-releasing channels. The data for most species suggests that IP3 mediates calcium release in eggs. Ryanodine receptors, which are the predominant calcium-releasing channel of the sarcoplasmic reticulum in muscle, have been reported to contribute to calcium release in eggs of some species. Agents that imitate the opening of the ryanodine receptor can, in some cases, initiate calcium release in the egg.

Inositol Trisphosphate
The second messenger inositol 1,4,5-trisphosphate (IP3) is generated following stimulation of membrane receptors coupled to heterotrimeric G proteins or tyrosine kinaselinked receptors. IP3 is the product of PLC-induced hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) which also produces sn-1,2diacylglycerol. The rst indication that there might be inositol phospholipid turnover was the detection of an increase in PIP2 and its precursor phosphatidylinositol monophosphate (PIP) within a minute after fertilization of
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Egg Activation

However, the physiological signicance of articially releasing calcium through the ryanodine receptor in eggs is unclear. For example, a variety of inhibitors against key components of ryanodine release show that ryanodinemediated calcium release is not required during fertilization of sea urchin eggs. In those species with ryanodine receptors, ryanodine receptor-mediated release could play an accessory role by amplifying calcium release once it is initiated by IP3. The calcium storage and releasing system in some eggs is modied during oocyte maturation in some species. Immature oocytes of starsh, hamster and mouse are less sensitive to IP3-induced calcium release than mature eggs (the stage at which fertilization normally occurs). Furthermore, when fertilized, immature oocytes release substantially less calcium than mature eggs. Enhanced calcium release in the egg after maturation may depend on a structural reorganization of the ER during maturation, as well as an increase in IP3 receptor number. Following mouse oocyte maturation, large ER clusters appear in the cortex of the mature egg. There is also an increase in immunoreactive mass of the type 1 IP3 receptor protein, suggesting that an increase in IP3 receptor number could also contribute to enhanced calcium release in the mature egg. Thus, during oocyte maturation, the calcium storage and releasing system is developed to permit the large increase in intracellular calcium needed at fertilization.

Calcium Waves
At fertilization, intracellular calcium rises rst at the site of sperm entry and then a wave of elevated calcium travels through the egg cytoplasm (Figure 1). The wave front passes through the cytoplasm at a velocity of 530 mm s 2 1 depending on the species and temperature. Waves are propagated by diusion of calcium and, to some extent, by

IP3, which promotes calcium release from the ER. The primary mechanism for wave propagation in most species is probably a form of calcium-induced calcium release in which calcium sensitizes IP3-mediated calcium release. A single calcium wave is triggered at fertilization in echinoderm, cnidarian, sh and frog eggs. In mammals and several other species, the rst calcium wave is followed by repetitive transients or spikes (Figure 2). Calcium oscillations have been reported to occur in fertilized eggs of all mammalian species so far investigated. The function of these oscillations after fertilization is not entirely known; however, oscillations persist until pronuclear formation in mouse embryos. It has been proposed that calcium oscillations may be necessary to release the egg from metaphase arrest and to fully inactivate maturationpromoting factor (MPF) and cytostatic factor (CSF). MPF and CSF together maintain meiotic arrest at metaphase II, and calcium-dependent inactivation of MPF and CSF is necessary for resumption of the cell cycle. In contrast to echinoderm and frog, in which a single calcium wave is sucient to activate the egg, the transition to interphase is much longer in mammals. Oscillations may be necessary in mammalians and some other species to provide a long-lasting period of calcium stimulation for complete activation. Repetitive transients can provide a sustained calcium signal without the cellular damage that would be caused by a prolonged period of elevated calcium. Calcium oscillations in mouse and hamster eggs are dependent on extracellular calcium. Evidence indicates that the rst calcium wave at fertilization depletes an intracellular calcium store and triggers capacitative calcium entry. The persistent inux of calcium is responsible for the repetitive calcium transients in the egg, which propagate through the cytoplasm in a wave-like manner. In mouse eggs, irrespective of the site of sperm entry, the repetitive waves begin in the cortex in the hemisphere opposite the meiotic spindle (vegetal pole). This region of

Figure 1 The calcium wave at fertilization of the frog egg is prevented by inhibition of IP3-induced calcium release. (a) Confocal images of the normal calcium wave in a fertilized Xenopus laevis egg that was previously injected with the calcium indicator Ca green dextran. (b) Images of a fertilized egg that was previously injected with Ca green dextran and a function-blocking antibody to the IP3 receptor. Although the calcium wave is inhibited, small spots of localized calcium release are detected in some eggs, as shown in (b). These hot spots may be localized calcium rises at the site of sperm egg fusion which are not completely blocked and are observed with other agents that inhibit the calcium wave (Glahn et al., 1999). Images in (a) and (b) are shown at 40-s intervals. From Runft et al. (1999), with permission.

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Egg Activation

Fluorescence

Sperm 0 0 40 Time (min)


Figure 2 Sperm-induced calcium oscillations in the mammalian egg. Changes in intracellular calcium were monitored with the fluorescent calcium indicator Fura-dextran. The fluorescence emission signal is displayed as the ratio of fluorescence for the 350-nm/385-nm excitation wavelengths. An increase in the fluorescence ratio represents an increase in intracellular free calcium.

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the cortex contains abundant ER, which functions as a stable cortical pacemaker responsible for generating calcium waves.

Proposed Mechanisms for Initiation of Egg Activation: PLCc and Tyrosine Kinase
Several studies have focused on whether PLCb or PLCg is activated at fertilization to cause the production of IP3 and release of calcium from intracellular stores. PLCb is activated by G proteins, while PLCg is activated by tyrosine kinases. Both PLCb and PLCg pathways are present in eggs of at least some species. Less is known about PLCd, the third isoform of PLC; its regulation is less well understood and it is not known if it functions in eggs. Evidence now suggests that calcium release in echinoderm eggs (starsh and sea urchin) requires the production of IP3 following the activation of the g isoform of phospholipase C (PLCg). The activity of PLCg increases after its two Src homology 2 (SH2) domains bind to an activated receptor or cytosolic tyrosine kinase. Experiments rst demonstrated that PLCg activation was required for fertilization and, more recently, evidence for the role of a tyrosine kinase has been obtained.
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Proteins containing the PLCg SH2 domains have been used to competitively inhibit PLCg activation by sequestering proteins that would normally interact with PLCg. Injection of a glutathione-S-transferase (GST) fusion protein containing the two SH2 domains of PLCg specically inhibits sperm-induced calcium release at fertilization of starsh and sea urchin eggs (Carroll et al., 1999). Activation of PLCg is probably caused by a tyrosine kinase. Tyrosine kinase activity increases within 15 s of insemination of sea urchin and starsh eggs. Evidence now indicates that an Src family kinase is required for fertilization of starsh eggs (Giusti et al., 1999). The participation of tyrosine kinases and PLCg in egg activation has also been examined in frog and mouse eggs. Experiments using SH2 domain injection to inhibit PLCg activation suggest that PLCg is not involved in mouse (Mehlmann et al., 1998) nor in frog (Runft et al., 1999) egg activation. However, a role for tyrosine kinase is suggested for frog egg activation, as an Src-related tyrosine kinase is activated within 1 min of fertilization and tyrosine kinase inhibitors prevent calcium release and egg activation at fertilization (Sato et al., 1999). The mechanism by which tyrosine activity is involved in initiating calcium release, if not through SH2 domain-mediated activation of PLCg (assuming the SH2 domain inhibitor is targeting PLCg), remains to be determined. G protein-mediated calcium release through activation of PLCb in frog and mouse eggs was suggested by experiments in which calcium waves were initiated after introduction of exogenous G protein-linked receptors. Certainly, a functioning G protein/PLCb pathway is present in eggs; however, a number of experiments using G protein antibodies indicate that the sperm-induced calcium rise in mouse and frog eggs is not mediated by heterotrimeric G proteins (Runft et al., 1999). Thus a role for PLCg or PLCb in initiating calcium release in vertebrate eggs is unclear, at least when examined using experiments based on the best-characterized mechanisms for activating these enzymes. Alternative mechanisms that would activate PLCg, PLCb, or the less well studied PLCd isoform at fertilization are possible, but not yet dened.

Proposed Mechanisms for Initiation of Egg Activation: Sperm-borne Activating Factors


It has been proposed that calcium release and subsequent activation of the egg are initiated by a sperm factor, which is introduced into the egg following spermegg fusion. Such a factor might be present in the sperm membrane and diuse to the egg membrane after the establishment of membrane continuity. An activated G protein or PLC in

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Egg Activation

the sperm membrane could conceivably activate a signalling cascade in the egg. However, there is as yet no evidence for this type of mechanism. Most hypotheses have centred on the possibility that a soluble cytosolic factor or some other relatively insoluble factor in the sperm is introduced into the egg cytoplasm after spermegg membrane fusion. It was suggested that calcium or IP3 could serve as an activating agent and be introduced into the cytoplasm after formation of a conduit between sperm and egg cytoplasm. However, at least for mammals, injection of neither calcium nor IP3 can replicate the normal series of calcium oscillations seen in the mammalian egg. Attention has focused on the idea that a soluble protein is introduced after fusion, as injection of protein-containing sperm extracts causes repetitive calcium transients much like those triggered by fertilization. Intracytoplasmic sperm injection experiments have suggested that the sperm-borne activating factor is associated with a more insoluble material that surrounds the sperms nucleus. The soluble protein factor (or factors) from mammalian sperm extracts that triggers calcium release is not speciesspecic. Extracts of mammalian sperm, including those made from hamster, human and pig sperm, activate eggs of the mouse as well as eggs of the same species. Among the invertebrate species, calcium oscillations are induced in nemertean eggs by sperm extract of the same species. Calcium release is promoted in ascidian eggs by human sperm extract, as well as ascidian sperm extract. Attempts have been made to purify the protein in the mammalian sperm extract, including isoelectric focusing and a series of chromatographic columns; however, the identity of this factor remains unknown. It has been proposed that a truncated form of the c-kit receptor in mouse sperm might interact with egg PLCg and cause activation. However, truncated c-kit has not yet been shown to release intracellular calcium and normal fertilization is not blocked by SH2 domain injection experiments. Recently, attention has been paid to the idea that the sperm may introduce PLC, rather than egg PLC being activated by some other sperm-borne factor. A fraction from cytosolic extracts of porcine sperm, which causes calcium release in both sea urchin egg homogenates and mouse eggs, has PLC activity (Parrington et al., 1999). This observation suggests an activation sequence in which PLC from the sperm is introduced into the egg and IP3 is produced through the action of this sperm-borne factor. The success of intracytoplasmic sperm injection in human-assisted reproduction and in several animal models forced further thought and experimentation to explain how the sperm activates the egg by its presence in the cytoplasm after bypassing the natural interactions at the egg membrane. In several studies, the sperm-borne activating factor was found to be localized in sperm material associated with the cytoplasmic face of the nucleus. Mouse egg activation (assessed by pronuclear formation) is triggered by injection of demembranated,

washed sperm, but only if the perinuclear material remains around the nucleus (Kimura et al., 1998). Recent experiments indicate that the perinuclear material initiates calcium oscillations. Moreover, egg activation is not induced by injection of round mouse spermatids, in which the perinuclear material is not yet assembled. Interestingly, full embryonic development can be initiated if round spermatids are injected along with sperm extract (Sakurai et al., 1999). The relationship between the perinuclear material and the soluble factor that activates eggs is unknown. The methods by which the factors are obtained suggest that they may be dierent proteins, but this has not been determined unequivocally.

Summary
Egg activation is triggered by an increase in intracellular calcium within the egg cytoplasm. The mechanism by which sperm initiate this calcium rise is not completely known, although IP3-mediated calcium release from the ER is required in most cases. A PLCg/tyrosine kinase pathway may be activated in echinoderms. Mammalian eggs and perhaps other vertebrate and invertebrate eggs may be activated by a soluble or a perinuclear factor released by sperm after spermegg fusion. Current research is aimed at determining the biochemical nature of these factors. A single factor may be sucient to activate the egg, but multiple pathways, which would help to ensure complete activation, have not been ruled out.

References
Carroll DJ, Albay DT, Terasaki M, Jae LA and Foltz KR (1999) Identication of PLCg-dependent and -independent events during fertilization of sea urchin eggs. Developmental Biology 206: 232247. Giusti AF, Carroll DJ, Abassi YA and Foltz KR (1999) Evidence that a starsh egg Src family tyrosine kinase associates with PLC-g1 SH2 domains at fertilization. Developmental Biology 208: 189199. Kimura Y, Yanagimachi R, Kuretake S et al. (1998) Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biology of Reproduction 58: 14071415. Mehlmann LM, Carpenter G, Rhee SG and Jae LA (1998) SH2 domain-mediated activation of phospholipase Cg is not required to initiate Ca2 1 release at fertilization of mouse eggs. Developmental Biology 203: 221232. Parrington J, Jones KT, Lai FA and Swann K (1999) The soluble sperm factor that causes Ca2 1 release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2 1 oscillations after injection into mouse eggs. Biochemical Journal 341: 14. Runft LL, Watras J and Jae LA (1999) Calcium release at fertilization of Xenopus eggs requires type I IP3 receptors, but not SH2 domainmediated activation of PLCg or Gq-mediated activation of PLCb. Developmental Biology 214: 399411. Sakurai A, Oda S, Kuwabara Y and Miyazaki S (1999) Fertilization, embryonic development, and ospring from mouse eggs injected with round spermatids combined with Ca2 1 oscillation-inducing sperm factor. Molecular Human Reproduction 5: 132138.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net

Egg Activation

Sato Y, Miyazaki S, Shikano T et al. (1998) Adenophostin, a potent agonist of the inositol 1,4,5-trisphosphate receptor, is useful for fertilization of mouse oocytes injected with round spermatids leading to normal ospring. Biology of Reproduction 58: 867873. Sato K, Iwao Y, Fujimura T et al. (1999) Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation. Developmental Biology 209: 308320. Terasaki M and Sardet C (1991) Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum. Journal of Cell Biology 115: 10311037.

Further Reading
Glahn D, Mark SD, Behr RK and Nuccitelli R (1999) Tyrosine kinase inhibitors block sperm-induced egg activation in Xenopus laevis. Developmental Biology 205: 171180. Jones KT (1998) Ca2 1 oscillations in the activation of the egg and development of the embryo in mammals. International Journal of Developmental Biology 42: 110. Kline D, Mehlmann LM, Fox C and Terasaki M (1999) The cortical endoplasmic reticulum (ER) of the mouse egg: localization of ER clusters in relation to the generation of repetitive calcium waves. Developmental Biology 215: 431442.

Lee SJ and Shen SS (1998) The calcium transient in sea urchin eggs during fertilization requires the production of inositol 1,4,5-trisphosphate. Developmental Biology 193: 195208. Miyazaki S, Shirakawa H, Nakada K and Honda Y (1993) Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2 1 release channel in Ca2 1 waves and Ca2 1 oscillations at fertilization of mammalian eggs. Developmental Biology 158: 6278. Palermo GD, Avrech OM, Colombero LT et al. (1997) Human sperm cytosolic factor triggers Ca2 1 oscillations and overcomes activation failure of mammalian oocytes. Molecular Human Reproduction 3: 367374. Rongish BJ, Wu W and Kinsey WH (1999) Fertilization-induced activation of phospholipase C in the sea urchin egg. Developmental Biology 215: 147154. Stricker SA (1999) Comparative biology of calcium signaling during fertilization and egg activation in animals. Developmental Biology 211: 157176. Swann K and Parrington J (1999) Mechanism of Ca2 1 release at fertilization in mammals. Journal of Experimental Zoology 285: 267 275. Williams CJ, Mehlmann LM, Jae LA, Kopf GS and Schultz RM (1998) Evidence that Gq family G proteins do not function in mouse egg activation at fertilization. Developmental Biology 198: 116127.

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