Calcium signaling at fertilization Sheldon S. Shen Iowa State University, Ames, Iowa, USA Calcium is well established as a second messenger in a diverse array of cell activities. Changes in intracellular Ca2+ activities range from localized releases to complex oscillations, which may encode specific cellular signals. The full variety of calcium responses is observed during the fertilization of different animal oocytes and eggs. Current research has focused on the cellular mechanisms that generate these Ca2+-activity changes.
Current Opinion in Genetics and Development 1992, 2:642-646 Introduction
mechanisms generate the observed natural responses still remains elusive.
The importance of changes in intracellular Ca 2 + activities as a regulatory agent in non-muscle cells was established by Steinhardt and colleagues [1-5] in a series of experiments using the fertilization reaction in sea-urchin eggs. They initially demonstrated that raising intracellular Ca 2 + activity with the ionophore A23187 was sufficient for parthenogenetic activation of animal eggs [1,2]. Then using first aequorin [3] and later fura-2 [4], they reported changes in Ca 2 + activity during fe~lization and subsequent cell division. Finally, by injecting a Ca 2 + chelator they prevented developmental events during fertilization and thus demonstrated that a rise in Ca 2 + is necessary for development [5]. Others have elegantly corroborated and extended these observations to demonstrate the necessity of Ca 2+ activity for fertilization in a wide variety of other organisms as well as in many somatic cellularactivation events [6°]. Since the initial simple observation of a transient rise (60-120s) in Ca 2+ activity from ~0.1 to ~2p.M in the sea-urchin egg, diverse changes during fertilization have been noted. These extend from localized Ca 2 + increases during polyspermy in Pleurodeles [7] to repetitive Ca 2+ transients during mammalian fertilization [8,9,10"]. This variety of Ca 2+ responses reflects the scope of Ca 2 + changes reported in somatic cells and may be important for determining the regulation of a broad range of Ca2+-dependent cellular events [11,-13-]. Several models of Ca2+ mechanisms for generating the different patterns of Ca 2 + changes have been proposed [14-17] and these mechanisms have generally involved inositol 1,4,5trisphosphate (InsP3)-induced Ca 2+ release and Ca 2+induced Ca 2+ release (CICR). Because of their size, eggs offer an ideal system for studying temporal and spatial interactions between different Ca 2+ regulatory mechanisms to generate Ca 2 + responses. Perhaps underscoring the importance of Ca 2 + as a regulatory messenger, animal eggs apparently have different arrangements of regulatory Ca2+-release mechanisms; but how these
InsP3-induced Ca 2+ release The principal source of Ca a+ during fertilization and parthenogenetic activation appears to be intracellular. Because animal sperm-activation requires external Ca 2+ , it has been difficult to explicitly demonstrate the independence from Ca 2 + irfflux of the Ca 2 + response in eggs during fertilization, but there is no doubt that eggs like most cells contain an intracellular Ca 2+ store. In all animal egg cells where it has been studied, Ca 2+release may be induced by InsP 3 (reviewed in [18"]). These observations suggested an analogy with somatic cell activation of a sperm-triggered inositol phospholipid hydrolysis during fertilization [19]. Unfortunately only in sea-urchin eggs [20,21.] and Xenopus oocytes [22.], has inositol phospholipid hydrolysis been studied. While several inositol phosphates may act as Ca 2+release agonists, InsP 3 is by 20-fold the most potent [23,24"]; however, other inositol phosphates, especially inositol 1,3,4,5-tetrakisphosphate, may have physiological functions [24.]. The InsPyinduced Ca 2 + -response differs amongst different al~nal eggs. In Xenopua oocytes, the spatial pattern of the InsPygenerated Ca2+-response by receptor activation as viewed by confocal microscopy, may propagate either as a planar or a circular wave [25"]. Furthermore, different receptor-coupled Ca2+-responses can be spatially and temporally distinguished [26-]. Similar spiral waves of Ca 2+ releases are produced by direct introduction of InsP 3 into the oocytes [27-.]. In sea-urchin eggs viewed by confocal microscopy, injection of InsP 3 induced a rapid planar wave of Ca 2 + elevation with an enllanced pronuclear response (SS Shen, unpublished data), which was similar to the Ca 2+ rise observed during fertilization [28-]. The InsPyinduced Ca 2+ increase is more rapid than that occurring during fertil-
Abbreviations cADPR--cyclic-ADP ribose; cGMP--cyclic-GMP; CICR--Ca2+-induced Ca2+ release; Dil--dicarbocyanine dye; ER-~endoplasmic reticulum; InsP3--inositol 1,4,5-triphosphate. 642
(~ Current Biology Ltd ISSN 0959-437X
Calcium signaling at fertilization Shen 643 ization and neither of the Ca 2+ responses are uniform through the egg [28.,29"]. Hamster and mouse oocytes both generate Ca2+ oscillations in response to InsP 3 injections [10",30]. These InsP3-induced oscillations do not continue for as long as the Ca 2+ oscillations during fertilization, which apparently required continued Ca 2+ influx.
release, but dithiothreitol blocks the action of thimerosal. As heparin does not block the Ca 2 + release induced by thimerosal [47"], the effect may be by increasing sensitivity of ClCR. A similar increase in sensitivity of ClCR may occur during fertilization [44].
Ca 2 +-induced Ca 2 + release
A variety of other agents has also been reported to act on Ca 2 + release in eggs. Of special interest is Ca 2 + release precipitated by cyclic-GMP (cGMP) [48-] and cyclic-ADP ribose (cADPR) [49] in intact sea-urchin eggs. Treatment of egg homogenates or injection of nanomolar concentrations of cADPR have been shown to induce rapid heparin-insensitive Ca 2+ release. The natural enzyme for cADPR generation is found in sea-urchin eggs and various mammalian tissues [50], but changes in endogenous levels of cADPR have not been determined. Interestingly cADPR does not affect Ca 2+ release in Xenopus oocytes (WB Busa, personal communication) or hamster eggs (K Swann, personal communication), although it may act to release Ca 2+ in rat pituitary cells [51]. It has been proposed that cADPR acts as the natural modulator of CICR in sea-urchin eggs [41"] and that its lack of effect in other animal eggs may be a consequence of an absence of ClCR (in Xenopus) or a different ClCR mechanism (in mammals). Injection of micromolar concentrations of cGMP induced a fertilization-like transient rise in Ca 2+ after a latency of at least 15s [48"]. The action of cGMP appears to be indirect, as it fails to release Ca 2+ in digitonin-permeabilized eggs. Furthermore, cGMP action is not heparin-sensitive and appears to stimulate Ca 2+ release through an unidentiffed InsPyindependent pathway [48.]. Delayed Ca 2+ release by cGMP has also been reported in Medaka eggs [52] and cGMP injection mimicked acetylcholineinduced membrane currents in Xenopus oocytes [53].
It has been suggested that a second Ca 2+-release mechanism of CICR, which is modeled after the mechanism active in muscle cells, occurs in eggs during fertilization [31]. This InsPyinsensitive CICR is characterized by sensitivity to caffeine and ryanodine, and inhibition by ruthenium red [32]. Although ClCR was originally proposed for Xenopus oocytes [31] and supported by observations that the Ca 2 + wave-propagation velocity is similar to the rate of Ca 2+ diffusion in the cells [25.], it appears that the oocytes lack ClCR. Rather, InsP 3 is absolutely required for generation of the Ca 2+ wave and heparin, an inhibitor of InsPyinduced Ca 2+ release [33], blocks Ca 2+ wave propagation [34"]. Rather than acting through CICR, Ca 2 + stimulated sensitization of the InsP 3 receptor [27"]. Absence of the CICR mechanism in Xenopus oocytes is further substantiated by reports that caffeine not only fails to induce Ca 2 + release, but inhibits the action of InsP 3 [33,34"]. In contrast, the seaurchin egg does contain a ClCR-like mechanism. While heparin blocked InsPyinduced Ca 2+ responses, it failed to block the Ca x+ rise during fertilization [37,38]. Caffeine and ryanodine induced Ca 2+ release in intact eggs, which was partially sensitive to ruthenium red [39",40]. Heparin has also been reported to block ryanodine action in isolated cortices preparation of Paracentrotus [40]; however, we have observed enhanced ryanodineinduced Ca 2+ release in heparin-loaded Lytechinus eggs [39"]. Antagonists of ClCR appear to have different activities in intact eggs and egg fractions. For example, ruthenium red at 30 I.tM has been reported to block caffeine and ryanodine action [41"], and InsP 3 action [42] in egg homogenates, but we have found only partial inhibition of Ca 2 + release by ryanodine and no effect on Ca 2 + release by caffeine or InsP 3 in the intact egg [39"]. Regardless of the differences in action of CICR antagonists, the ClCR mechanism active in sea-urchin eggs differs from that described in muscle and most somatic cells. Sudden changes in Ca 2+ fail to elicit CICR [29"] and agonist responses are graded, such that caffeine and ryanodine have synergistic actions [39*]. The absence of direct Ca 2 + action may be the result of the high Ca 2 + buffer capacity of the cytosol [29",43]; however, ClCR is observed in sea-urchin egg homogenate preparations only after excess Ca 2+ loading of the microsomes [41.]. A ClCR mechanism has also been suggested in hamster [44] and mouse [45] oocytes. Ca 2+ release in mammalian oocytes is neither caffeine nor ryanodine sensitive, but thimerosal, a sulphydryl reagent, causes Ca 2 + oscillations similar to those seen at fertilization and enhances the sensitivity of eggs to injections of Ca 2+ [46.]. How thimerosal acts is unclear as other sulphydryl reagents do not induce Ca 2 +
cGMP and cADPR-induced Ca 2+ release
Localization of Ca2+-release mechanisms Understanding the mechanism of Ca 2+ release during fertilization requires information on the distribution of the different Ca2+-release mechanisms. For example, it is unclear whether eggs contain separate Ca 2+ stores with different release mechanisms, or a single store with multiple Ca 2+ -release mechanisms, or a condition inclusive of both possibilities, which has been reported in Purkinje cells [54]. Ryanodine receptors have been reported to immuno!ocalize to the cortex in sea-urchin eggs and form a reticulum that is associated with cortical granules and the plasma membrane [55"]. This region corresponds to that to which the calsequestrinlike protein is localized [56"] and is also InsPysensitive [57"]. In a study using the diffusion of dicarbocyanine dye (Dil), the endoplasmic reticulum (ER) of sea-urchin eggs has been suggested to be a cellwide interconnected compartment [58"], although it is possible that some other Ca2+-regulatory regions are discontinuous with the Dil-stained ER. An InsPyindependent cADPR-specific receptor associated with Ca 2 +storing microsomes from sea-urchin eggs has been reported [59"], but its distribution throughout the egg
644
Pattern formation' and developmental mechanisms is u n k n o w n . F u r t h e r e v i d e n c e for a single Ca 2+ store in the sea-urchin egg was p r o v i d e d by the a b s e n c e o f Ca 2+ release i n d u c e d by the i o n o p h o r e A23187 after Ca 2+ release i n d u c e d by a p o o r l y m e t a b o l i z e d InsP 3 analog [48.], H o w e v e r , h y p e r t o n i c sea w a t e r released Ca 2+ in fertilized eggs, w h i c h was u n r e s p o n sive to i o n o p h o r e A23187 t r e a t m e n t [60]. A m o r e c o m p l e x regulation o f Ca 2+ s t o r e is s u g g e s t e d by cADPR-induced Ca 2 + release in thapsigargin-treated eggs after I n s P y i n d u c e d Ca 2+ release (SS Shen, u n p u b lished data). Thapsigargin is an i n h i b i t o r o f ER Ca 2+ ATPase [61] and b l o c k s Ca 2 + s e q u e s t r a t i o n in sea-urchin h o m o g e n a t e s (SS Shen, u n p u b l i s h e d data).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1. STEINHAP, DT R, EPEI. D: Activation of Sea Urchin Eggs by a Calcium ionophore. Proc Naa Acx+d Sci U S A 1974, 71:1915-1919. 2.
STEINHARDTRA, EPEL D, CARROLLES, YANAGANIACHIR: Is Calcium Ionophore a Universal Activator for Unfertilized Eggs? Nature 1974, 252:41-43.
3.
STEINHARDTRA, ZUCKER R, SCHATI~N G: Intracellular Calcium Release at Fertilization in the Sea Urchin Egg. Dev Biol
1977, 58:185-196.
How does sperm trigger
Ca 2 +
release?
Perhaps the m o s t intriguing q u e s t i o n associated with fertilization is h o w s p e r m triggers the Ca 2+ release. Evidence for InsP3-sensitive Ca 2+ release and analogy with somatic cell-activation has s u g g e s t e d s p e m a interaction with a r e c e p t o r and G - p r o t e i n - c o u p l e d p h o s p h o lipase-C [19]. E x p e r i m e n t a l a n o m a l i e s d u r i n g sea-urchin and m a m m a l i a n fertilization have cast s o m e d o u b t s o n the i m p o r t a n c e o f inositol p h o s p h o l i p i d hydrolysis during fertilization ( r e v i e w e d in [18"'] ). An earlier h y p o t h e sis that the s p e r m carries an activating factor, w h i c h sets off the c h a n g e in Ca 2+ [62], is again favored. A stud), using electrical m e a s u r e m e n t s d u r i n g fertilization o f seaurchin eggs, has s h o w n that the continuity o f s p e r m and egg c y t o p l a s m is a p p a r e n d y r e q u i r e d for egg activation [63"']. T h e r e are a n u m b e r o f putative activating factors ( r e v i e w e d in [ 1 8 . . ] ) , b u t the m o s t p r o m i s i n g a p p e a r s to b e a p r o t e i n factor in h a m s t e r s p e r m , w h i c h triggers fertilization-like Ca 2+ oscillations in h a m s t e r eggs [64], m o u s e eggs and n e u r o n s (K Swarm, p e r s o n a l c o m m u n i cation). A s o l u b l e s p e m a factor has also b e e n r e p o r t e d for sea-urchin fertilization [65].
Conclusion T h e rise in Ca 2 + activity d u r i n g fertilization is u n d o u b t edly r e q u i r e d for activation o f the egg, but the bases o f the Ca 2+ c h a n g e s r e m a i n unclear. C u r r e n t r e s e a r c h suggests the different Ca 2+ signaling o b s e r v e d d u r i n g fertilization is the result o f different Ca 2 + -release m e c h a n i s m s p r e s e n t in eggs. T h e c o n t r o l a n d interactions o f t h e s e m e c h a n i s m s a p p e a r m o r e c o m p l e x than initially hypothesized. F u r t h e r e x p e r i m e n t s are r e q u i r e d b o t h to clarify regulation o f t h e s e C a 2 + - r e l e a s e m e c h a n i s m s , and h o w s p e r m initiates Ca 2 + signaling d u r i n g fertilization.
Acknowledgements 1 thank Drs K Campbell, D Clapham, R Nuccitelli, G Schatten, K Swarm and M Whitaker for sharing their manuscripts and thoughts on this subject. Work in my laboratory has been supported by National Science Foundation DCB 89-03837.
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645
646
Patternformation and developmental mechanisms CICR in a sea-urchin egg homogenate preparation is described. The authors suggest that cADPR may be the natural agonist of ClCR during sea-urchin fertilization. This provocative idea is marred by several experiment procedural ditficulties, which are not described in the article. For example, the authors use ruthenium red, which significandy quenches fluorescence signals, which prevents accurate measurement of Ca 2+ changes. 42.
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CARROLLJ, SWANN K: Spontaneous Cytosolic Calcium OsciUations Driven by Inositol Trisphosphate O c c u r during in Vitro Maturation of Mouse Oocytes. J Biol Chem 1992, 267:11196-11201. Heparin injection blocks lnsPyinduced Ca 2 + oscillations, but does not affect thimerosal actions on CICR. 48.
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RUSINKON, LEE HC: Widespread O c c u r r e n c e in Animal Tissues of an Enzyme Catalyzing the Conversion of NAD + into a Cyclic Metabolite with Intracellular Ca2+-Mobilizing Activity. J Biol Cbem 1989, 264:11725-11731.
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KOSHIYAMAH, LEE HC, T&SHJIAN AH: Novel Mechanism of lntracellular Calcium Release in Pituitary Cells. J Biol Cbem 1991, 266:16985-16988.
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IW,V~IAI,'SUT, YOSHIMOTOY, HIRAMOTOY: Mechanism of Ca 2+ Release in M e d a k a Eggs Microinjected with Inositol 1,4,5Trisphosphate and Ca 2+. Dev Biol 1988, 129:191-197.
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WALTON PD, AIREY JA, Su'rKo JL, BECK CF, MIGNERY FA, S0DHOF TC, DEEP,NICK TJ, ELLISMANMH: Ryanodine and Inositol Trisphosphate Receptors Coexist in Avian Cerebellar Purkinje Neurons. J Cell Biol 1991, 113:1145-1157.
55.
MCPHERSONSM, MCPHEP~SON PS, MATHEWS L, CAbIPBELL KP, LONGOFJ: Cortical Localization of a Calcium Release Channel in Sea Urchin Eggs. J Cell Biol 1992, 116:1111-1121. In this study, ryanodine receptors are localized to the egg cortex by using polyclonal antibodies to striated muscle t3,anodine receptors. An ~380 kD protein is identified using immunoblot analysis. Immunoreactivity of file cortex is altered by treatment of corticies with caffeine, ryanodine, Ca 2+ or A23187. •
56. .
TERAS/,.KIM, HENSON J, BEGG D, KAMINERB, SARDET C: Characterization of Sea Urchin Egg Endoplasmic Reticulum in Cortical Preparations. Dev Biol 1991, 148:398--401. In this work, file cortical ER is stained by Dil and observed to colocalized with calsequestrin-like protein. The cortical ER often encircles cortical granules, and is thus apparently positioned for initiating cortical granule fusion and propagating the Ca 2+ wave. 57. •
TERASAKIM, SARDET C: Demonstration of Calcium Uptake and Release by Sea Urchin Egg Cortical Endoplasmic Reticulum. J Cell Biol 1991, 115:1031-1037. The ER of isolated Arbacia, but not other species of sea urchin, may be loaded with fluo-3. The cortical ER is shown to be a site of ATPdependent Ca 2 + sequestration and lnsPyinduced Ca 2 + release. 58. •
TERA~,AKIM, JAFFE LA: Organization of the Sea Urchin Egg Endoplasmic Reticulum and its Reorganization at Fertilization. J Cell Biol 1991, 114:929-940. The ER of intact eggs is stained with Dil and the application of confocal microscopy reveals an extensive cell-wide, interconnected comparmlent that is in continuous motion. Tile ER became reorganized coincident with the Ca 2 + wave at fertilization, but by 5--8 rain it had returned to an organization nomlally seen in unfertilized eggs. A possible role for ER reorganization and sperm pronuclear events is discussed. 59. .,
LEE HC: Specific Binding of Cyclic ADP-ribose to Calciumstoring Microsomes from Sea Urchin Eggs. J Biol Chem 1991, 266:2276--2281. Specific binding of cADPR to a saturatable site on the Ca 2+ -storing microsomes is reported. The binding is unaffected by NAD + , ADP-ribose, heparin and InsPy Tbe binding shows a pH optimum at about 6.7 and is Ca 2+ -sensitive, which differs from the binding of InsP3 to its receptor. 60.
ZUCKERRS, STEINHARDT RA, WINKLER MM: lntracellular Calcium and Mechanisms of Parthenogenetic Activation of Sea Urchin Eggs. De,, Biol 1978, 65:285-295.
61.
TH,vs'rRuI'O, CULLEN PJ, DROBAK BK, HANLEY MR, DAWSON AP: Thapsigargin, a T u m o r Promoter, Discharges Intracellular Ca 2+ Stores by Specific Inhibition of the Endoplasmic Reticulum Ca2+-ATPase. Proc Nail Acad Sci U S A 1990, 87:2466-2470.
62.
JAFFE LF: Sources of Calcium in Egg Activation: A Review. Det, Biol 1983, 99:256-276.
63. McCuU.OH DH, CHAMBERS EL: Fusion of Membranes during •• Fertilization. J Gen Physiol 1992, 99:137-175. Ea rly events of s p e m l - e g g interactions are ex:unined by capacitance and current m~k,;urements. These measurements suggest that egg activation requires sperm-egg fusion, which supports tile hYl)Othesis that egg activation is driven by a sperm cTtosolic factor. 64.
SW,~N K: A Cytosolic Sperm Factor Stimulates Repetitive Calcium Increases and Mimics Fertilization in Hamster Eggs. Det,elopment 1990, 110:1295-1302.
65.
DALE B, DEFEUCE LJ, EHRENSTEIN G: Injection of a Soluble Sperm Extract into Sea Urchin Eggs Triggers the Cortical Reaction. E.xperentia 1985, 41:1068-1070.
SS Shen, Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011, USA.
Current Opinion in Genetics and Development 1992, 2:642-646 Introduction
mechanisms generate the observed natural responses still remains elusive.
The importance of changes in intracellular Ca 2 + activities as a regulatory agent in non-muscle cells was established by Steinhardt and colleagues [1-5] in a series of experiments using the fertilization reaction in sea-urchin eggs. They initially demonstrated that raising intracellular Ca 2 + activity with the ionophore A23187 was sufficient for parthenogenetic activation of animal eggs [1,2]. Then using first aequorin [3] and later fura-2 [4], they reported changes in Ca 2 + activity during fe~lization and subsequent cell division. Finally, by injecting a Ca 2 + chelator they prevented developmental events during fertilization and thus demonstrated that a rise in Ca 2 + is necessary for development [5]. Others have elegantly corroborated and extended these observations to demonstrate the necessity of Ca 2+ activity for fertilization in a wide variety of other organisms as well as in many somatic cellularactivation events [6°]. Since the initial simple observation of a transient rise (60-120s) in Ca 2+ activity from ~0.1 to ~2p.M in the sea-urchin egg, diverse changes during fertilization have been noted. These extend from localized Ca 2 + increases during polyspermy in Pleurodeles [7] to repetitive Ca 2+ transients during mammalian fertilization [8,9,10"]. This variety of Ca 2+ responses reflects the scope of Ca 2 + changes reported in somatic cells and may be important for determining the regulation of a broad range of Ca2+-dependent cellular events [11,-13-]. Several models of Ca2+ mechanisms for generating the different patterns of Ca 2 + changes have been proposed [14-17] and these mechanisms have generally involved inositol 1,4,5trisphosphate (InsP3)-induced Ca 2+ release and Ca 2+induced Ca 2+ release (CICR). Because of their size, eggs offer an ideal system for studying temporal and spatial interactions between different Ca 2+ regulatory mechanisms to generate Ca 2 + responses. Perhaps underscoring the importance of Ca 2 + as a regulatory messenger, animal eggs apparently have different arrangements of regulatory Ca2+-release mechanisms; but how these
InsP3-induced Ca 2+ release The principal source of Ca a+ during fertilization and parthenogenetic activation appears to be intracellular. Because animal sperm-activation requires external Ca 2+ , it has been difficult to explicitly demonstrate the independence from Ca 2 + irfflux of the Ca 2 + response in eggs during fertilization, but there is no doubt that eggs like most cells contain an intracellular Ca 2+ store. In all animal egg cells where it has been studied, Ca 2+release may be induced by InsP 3 (reviewed in [18"]). These observations suggested an analogy with somatic cell activation of a sperm-triggered inositol phospholipid hydrolysis during fertilization [19]. Unfortunately only in sea-urchin eggs [20,21.] and Xenopus oocytes [22.], has inositol phospholipid hydrolysis been studied. While several inositol phosphates may act as Ca 2+release agonists, InsP 3 is by 20-fold the most potent [23,24"]; however, other inositol phosphates, especially inositol 1,3,4,5-tetrakisphosphate, may have physiological functions [24.]. The InsPyinduced Ca 2 + -response differs amongst different al~nal eggs. In Xenopua oocytes, the spatial pattern of the InsPygenerated Ca2+-response by receptor activation as viewed by confocal microscopy, may propagate either as a planar or a circular wave [25"]. Furthermore, different receptor-coupled Ca2+-responses can be spatially and temporally distinguished [26-]. Similar spiral waves of Ca 2+ releases are produced by direct introduction of InsP 3 into the oocytes [27-.]. In sea-urchin eggs viewed by confocal microscopy, injection of InsP 3 induced a rapid planar wave of Ca 2 + elevation with an enllanced pronuclear response (SS Shen, unpublished data), which was similar to the Ca 2+ rise observed during fertilization [28-]. The InsPyinduced Ca 2+ increase is more rapid than that occurring during fertil-
Abbreviations cADPR--cyclic-ADP ribose; cGMP--cyclic-GMP; CICR--Ca2+-induced Ca2+ release; Dil--dicarbocyanine dye; ER-~endoplasmic reticulum; InsP3--inositol 1,4,5-triphosphate. 642
(~ Current Biology Ltd ISSN 0959-437X
Calcium signaling at fertilization Shen 643 ization and neither of the Ca 2+ responses are uniform through the egg [28.,29"]. Hamster and mouse oocytes both generate Ca2+ oscillations in response to InsP 3 injections [10",30]. These InsP3-induced oscillations do not continue for as long as the Ca 2+ oscillations during fertilization, which apparently required continued Ca 2+ influx.
release, but dithiothreitol blocks the action of thimerosal. As heparin does not block the Ca 2 + release induced by thimerosal [47"], the effect may be by increasing sensitivity of ClCR. A similar increase in sensitivity of ClCR may occur during fertilization [44].
Ca 2 +-induced Ca 2 + release
A variety of other agents has also been reported to act on Ca 2 + release in eggs. Of special interest is Ca 2 + release precipitated by cyclic-GMP (cGMP) [48-] and cyclic-ADP ribose (cADPR) [49] in intact sea-urchin eggs. Treatment of egg homogenates or injection of nanomolar concentrations of cADPR have been shown to induce rapid heparin-insensitive Ca 2+ release. The natural enzyme for cADPR generation is found in sea-urchin eggs and various mammalian tissues [50], but changes in endogenous levels of cADPR have not been determined. Interestingly cADPR does not affect Ca 2+ release in Xenopus oocytes (WB Busa, personal communication) or hamster eggs (K Swann, personal communication), although it may act to release Ca 2+ in rat pituitary cells [51]. It has been proposed that cADPR acts as the natural modulator of CICR in sea-urchin eggs [41"] and that its lack of effect in other animal eggs may be a consequence of an absence of ClCR (in Xenopus) or a different ClCR mechanism (in mammals). Injection of micromolar concentrations of cGMP induced a fertilization-like transient rise in Ca 2+ after a latency of at least 15s [48"]. The action of cGMP appears to be indirect, as it fails to release Ca 2+ in digitonin-permeabilized eggs. Furthermore, cGMP action is not heparin-sensitive and appears to stimulate Ca 2+ release through an unidentiffed InsPyindependent pathway [48.]. Delayed Ca 2+ release by cGMP has also been reported in Medaka eggs [52] and cGMP injection mimicked acetylcholineinduced membrane currents in Xenopus oocytes [53].
It has been suggested that a second Ca 2+-release mechanism of CICR, which is modeled after the mechanism active in muscle cells, occurs in eggs during fertilization [31]. This InsPyinsensitive CICR is characterized by sensitivity to caffeine and ryanodine, and inhibition by ruthenium red [32]. Although ClCR was originally proposed for Xenopus oocytes [31] and supported by observations that the Ca 2 + wave-propagation velocity is similar to the rate of Ca 2+ diffusion in the cells [25.], it appears that the oocytes lack ClCR. Rather, InsP 3 is absolutely required for generation of the Ca 2+ wave and heparin, an inhibitor of InsPyinduced Ca 2+ release [33], blocks Ca 2+ wave propagation [34"]. Rather than acting through CICR, Ca 2 + stimulated sensitization of the InsP 3 receptor [27"]. Absence of the CICR mechanism in Xenopus oocytes is further substantiated by reports that caffeine not only fails to induce Ca 2 + release, but inhibits the action of InsP 3 [33,34"]. In contrast, the seaurchin egg does contain a ClCR-like mechanism. While heparin blocked InsPyinduced Ca 2+ responses, it failed to block the Ca x+ rise during fertilization [37,38]. Caffeine and ryanodine induced Ca 2+ release in intact eggs, which was partially sensitive to ruthenium red [39",40]. Heparin has also been reported to block ryanodine action in isolated cortices preparation of Paracentrotus [40]; however, we have observed enhanced ryanodineinduced Ca 2+ release in heparin-loaded Lytechinus eggs [39"]. Antagonists of ClCR appear to have different activities in intact eggs and egg fractions. For example, ruthenium red at 30 I.tM has been reported to block caffeine and ryanodine action [41"], and InsP 3 action [42] in egg homogenates, but we have found only partial inhibition of Ca 2 + release by ryanodine and no effect on Ca 2 + release by caffeine or InsP 3 in the intact egg [39"]. Regardless of the differences in action of CICR antagonists, the ClCR mechanism active in sea-urchin eggs differs from that described in muscle and most somatic cells. Sudden changes in Ca 2+ fail to elicit CICR [29"] and agonist responses are graded, such that caffeine and ryanodine have synergistic actions [39*]. The absence of direct Ca 2 + action may be the result of the high Ca 2 + buffer capacity of the cytosol [29",43]; however, ClCR is observed in sea-urchin egg homogenate preparations only after excess Ca 2+ loading of the microsomes [41.]. A ClCR mechanism has also been suggested in hamster [44] and mouse [45] oocytes. Ca 2+ release in mammalian oocytes is neither caffeine nor ryanodine sensitive, but thimerosal, a sulphydryl reagent, causes Ca 2 + oscillations similar to those seen at fertilization and enhances the sensitivity of eggs to injections of Ca 2+ [46.]. How thimerosal acts is unclear as other sulphydryl reagents do not induce Ca 2 +
cGMP and cADPR-induced Ca 2+ release
Localization of Ca2+-release mechanisms Understanding the mechanism of Ca 2+ release during fertilization requires information on the distribution of the different Ca2+-release mechanisms. For example, it is unclear whether eggs contain separate Ca 2+ stores with different release mechanisms, or a single store with multiple Ca 2+ -release mechanisms, or a condition inclusive of both possibilities, which has been reported in Purkinje cells [54]. Ryanodine receptors have been reported to immuno!ocalize to the cortex in sea-urchin eggs and form a reticulum that is associated with cortical granules and the plasma membrane [55"]. This region corresponds to that to which the calsequestrinlike protein is localized [56"] and is also InsPysensitive [57"]. In a study using the diffusion of dicarbocyanine dye (Dil), the endoplasmic reticulum (ER) of sea-urchin eggs has been suggested to be a cellwide interconnected compartment [58"], although it is possible that some other Ca2+-regulatory regions are discontinuous with the Dil-stained ER. An InsPyindependent cADPR-specific receptor associated with Ca 2 +storing microsomes from sea-urchin eggs has been reported [59"], but its distribution throughout the egg
644
Pattern formation' and developmental mechanisms is u n k n o w n . F u r t h e r e v i d e n c e for a single Ca 2+ store in the sea-urchin egg was p r o v i d e d by the a b s e n c e o f Ca 2+ release i n d u c e d by the i o n o p h o r e A23187 after Ca 2+ release i n d u c e d by a p o o r l y m e t a b o l i z e d InsP 3 analog [48.], H o w e v e r , h y p e r t o n i c sea w a t e r released Ca 2+ in fertilized eggs, w h i c h was u n r e s p o n sive to i o n o p h o r e A23187 t r e a t m e n t [60]. A m o r e c o m p l e x regulation o f Ca 2+ s t o r e is s u g g e s t e d by cADPR-induced Ca 2 + release in thapsigargin-treated eggs after I n s P y i n d u c e d Ca 2+ release (SS Shen, u n p u b lished data). Thapsigargin is an i n h i b i t o r o f ER Ca 2+ ATPase [61] and b l o c k s Ca 2 + s e q u e s t r a t i o n in sea-urchin h o m o g e n a t e s (SS Shen, u n p u b l i s h e d data).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1. STEINHAP, DT R, EPEI. D: Activation of Sea Urchin Eggs by a Calcium ionophore. Proc Naa Acx+d Sci U S A 1974, 71:1915-1919. 2.
STEINHARDTRA, EPEL D, CARROLLES, YANAGANIACHIR: Is Calcium Ionophore a Universal Activator for Unfertilized Eggs? Nature 1974, 252:41-43.
3.
STEINHARDTRA, ZUCKER R, SCHATI~N G: Intracellular Calcium Release at Fertilization in the Sea Urchin Egg. Dev Biol
1977, 58:185-196.
How does sperm trigger
Ca 2 +
release?
Perhaps the m o s t intriguing q u e s t i o n associated with fertilization is h o w s p e r m triggers the Ca 2+ release. Evidence for InsP3-sensitive Ca 2+ release and analogy with somatic cell-activation has s u g g e s t e d s p e m a interaction with a r e c e p t o r and G - p r o t e i n - c o u p l e d p h o s p h o lipase-C [19]. E x p e r i m e n t a l a n o m a l i e s d u r i n g sea-urchin and m a m m a l i a n fertilization have cast s o m e d o u b t s o n the i m p o r t a n c e o f inositol p h o s p h o l i p i d hydrolysis during fertilization ( r e v i e w e d in [18"'] ). An earlier h y p o t h e sis that the s p e r m carries an activating factor, w h i c h sets off the c h a n g e in Ca 2+ [62], is again favored. A stud), using electrical m e a s u r e m e n t s d u r i n g fertilization o f seaurchin eggs, has s h o w n that the continuity o f s p e r m and egg c y t o p l a s m is a p p a r e n d y r e q u i r e d for egg activation [63"']. T h e r e are a n u m b e r o f putative activating factors ( r e v i e w e d in [ 1 8 . . ] ) , b u t the m o s t p r o m i s i n g a p p e a r s to b e a p r o t e i n factor in h a m s t e r s p e r m , w h i c h triggers fertilization-like Ca 2+ oscillations in h a m s t e r eggs [64], m o u s e eggs and n e u r o n s (K Swarm, p e r s o n a l c o m m u n i cation). A s o l u b l e s p e m a factor has also b e e n r e p o r t e d for sea-urchin fertilization [65].
Conclusion T h e rise in Ca 2 + activity d u r i n g fertilization is u n d o u b t edly r e q u i r e d for activation o f the egg, but the bases o f the Ca 2+ c h a n g e s r e m a i n unclear. C u r r e n t r e s e a r c h suggests the different Ca 2+ signaling o b s e r v e d d u r i n g fertilization is the result o f different Ca 2 + -release m e c h a n i s m s p r e s e n t in eggs. T h e c o n t r o l a n d interactions o f t h e s e m e c h a n i s m s a p p e a r m o r e c o m p l e x than initially hypothesized. F u r t h e r e x p e r i m e n t s are r e q u i r e d b o t h to clarify regulation o f t h e s e C a 2 + - r e l e a s e m e c h a n i s m s , and h o w s p e r m initiates Ca 2 + signaling d u r i n g fertilization.
Acknowledgements 1 thank Drs K Campbell, D Clapham, R Nuccitelli, G Schatten, K Swarm and M Whitaker for sharing their manuscripts and thoughts on this subject. Work in my laboratory has been supported by National Science Foundation DCB 89-03837.
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KHNED, KUNEJT: Repetitive Calcium Transients and the Role of Calcium in Exocytosis and Cell Cycle Activation in the Mouse Egg. Dev Biol 1992, 149: 80-89. In this work, mouse eggs were loaded with different concentrations of the Ca2+ chelator BAPTA. Sperm entry occurred in all eggs regardless of BAPTAconcentration, but inhibition of both cortical granule exocytosis and resumption of the cell cycle correlated with BAPTA inhibition of the Ca2+ oscillations at fertilization. 12. •
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21. •
27. •.
LECHIJ-:rrF_R JD, CIAPIIAM DE: Molecular Mechanisms of Intracellular Calcium Excitability in X e n o p u s laevis Oocytes. Cell 1992, 69:283-294. It is shown that Ca 2+ wave propagation is absolutely dependent upon lnsP 3, and that Ca 2+ levels modulate InsPyreceptor activity. A model for Ca 2+ wave propagation is presented. Furthern3ore, it is demonstrafed that thapsigargin does not induce regenerative Ca 2+ activity, but after 4 h in a Ca2+ -free condition, the ooQ'te is unresponsive to InsP3, although caffeine does elicit a small Ca2 + increase. 28. •
S'rRfCKtJ:JtSA, CENTONZI:.VE, PADI)OCKSW, SCIIATI'FNG: Confocal Microscopy of Fertilization-induced Calcium Dynamics in Sea Urchin Eggs. Dev Biol 1992, 149:370-380. The fertilization-induced Ca 2 + rise is viewed by confocal microscopy. All enhanced nuclear Ca 2+ release is observed. Similar resuhs are also obtained with both fluo-3 and c'dcium green-loaded eggs. 29. ,
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31.
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24. •
PARKEa I, IvOItlb\ I: Inositol Tetrakisphosphate Liberates Stored Ca 2+ in X e n o p u s Oocytes and Facilitates Responses to Inositol Trisphosphate..I I"lo'siol (Lond) 1991, 433:207-227. Tim Caa+ -release actions of InsP3 rnetabolites are examined. A facilitatory role for inositol 1,3,4,5-temtkisphosphate is established. LE..ClILEITF~RJ, GII~,XRD S, PF.RALTA E, CIAI'I-I,VM D: Spiral Catcium Wave Propagation and Annihilation in X e n o p u s laevis Oocytes. Science 1991, 252:123-126. Both planar and circular propagation of Ca2 + waves cafl be ohserved using confocal microscopy following muscarinic acetylcholine receptor activation. The propagation velocity is equal to the rote of Ca 2 + diffusion, which supports CICR as the dominant mechanism for Ca 2 + wave propagation. This conclusion is rectified with the apparent absence of CICR in the work by Lechleiter and Calpham [27•°].
34. •
DELIsI.ES, WECSU, MJ: lnositol Trisphosphate is Required for the Propagation of Calcium Waves in X e n o p u s Oocytes../ Biol Chem 1992, 267:7963-7966. Heparin is shown to I-dock Ca 2 + WaVe propagation induced by lnsP 3 anti IP3S3, which suggests the critical importance of InsP3 rather than Ca 2+ for Ca 2+ release. Furthermore, localized increase in Ca 2+ without production of InsP3 does not tri,Kqer Ca 2 + waves. 35.
BERRIDGE ix'IJ: Caffeine Inhibits lnositol-trisphosphate-induced Membrane Potential Oscillations in XettOpllS Oocytes. Proc R Sr~: Lond /Biol/ 1991, 244:57--62.
36.
P,~aiKi:.8I, I\'ORmX I: C,'iffeine Inhibits lnositol TrisphosphateMediated Liberation of Intracellular Calcium in X e n o p u s Oocytes..I Phys&l (Lond) 1991, 433:229-240.
37.
RAKOWTL, SHEN SS: Multiple Stores of Calcium are Released in the Sea Urchin Egg during Fertilization. Proc Nail Acad Sci U S A 1990, 87:9285-9289.
38.
CROSSLEY 1, WHAHJ:.Y T, \',glIITAKER M: Guanosine 5"-Thiotriphosphate may Stimulate Phosphoinositide Messenger Production in Sea Urchin Eggs by a Different Route than the Fertilizing Sperm. Cell Reg 1991, 2:121-133.
25. •
26. •.
LECHLEITERJ, GIIbXRD S, Cb',dHIAM D, PEPaU.TA E: Subcellular Patterns of Calcium Release Determined by G ProteinSpecific Residues of Muscarinic Receptors. Natttre 1991, 350:505-508. Different Ca 2 + wave propagation occurred with activation of different suhtypes of muscarinic acetylcholine receptors. The spatial and temporal separation of the Ca 2 + -release patterns suggested they may encode distinct cell signaling pathways, which are mediatetl by different G proteins.
39. •
BUCKWR, RAKOWTL, StlI:.N SS: Synergistic Release of Calcium in Sea Urchin Eggs by Caffeine and Ryanodine. Exp Cell Res 1992, in press. Caffeine and t3,anodine are shown to stimulate graded anti synergistic Ca 2+ release, which su&Rests that dais release mechanism, unlike the CICR mechanisna described in somatic cells, is insensitive to sudden changes in Ca 2 +. Drag sensitivity differences to somatic CICR and Ca 2 + release in sea-urchin homogenates are also described. 40.
SARDI'.'TC, GILI.OT 1, Ruse1W.R & PAYANP, GIRARDJP, I)E RENZlS G: Ryanodine Activates Sea Urchin Eggs. Det, Growth Differ 1992, 34:37-42.
645
646
Patternformation and developmental mechanisms CICR in a sea-urchin egg homogenate preparation is described. The authors suggest that cADPR may be the natural agonist of ClCR during sea-urchin fertilization. This provocative idea is marred by several experiment procedural ditficulties, which are not described in the article. For example, the authors use ruthenium red, which significandy quenches fluorescence signals, which prevents accurate measurement of Ca 2+ changes. 42.
FUJIWARAA, TAGUCHI K, YASUM&SU 1: Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs K n o w n to Cause Ca 2+ Release from Isolated Sarcoplasmic Reticulum. Det, Growth Differ 1990, 32:303-314.
43.
SWANNK, WHrr,Ue,ER M: T h e Part Played by Inositol Triphosphate and Calcium in the Propagation of t h e Fertilization Wave in Sea Urchin Eggs. J Cell Biol 1986, 103:2333-2342.
44.
IGUSAY, MIYAZAKIS: Effects of Altered Extracellular and Intracellular Calcium Concentration on Hyperpolarizing Responses of Hamster Egg..I Physiol (Lond) 1983, 340:611~,32.
45.
PERESA: InsPy and Ca2+-Induced Ca 2+ Release in Single Mouse Oocytes. FEBS Left 1990, 275:213-216.
46. .
SWANNK: Thimerosal Causes Calcium Oscillations and Sensitizes Calcium-induced Calcium Release in UnfertiliTed Hamster Eggs. FEBS Lett 1991, 278:175-178. This work demonstrates that hanlster eggs are not responsive to caffeine, but that thimerosal, a sulfhydryl reagent, causes Ca 2+ release and enhances file sensitivity of eggs to Ca 2 + injections. Why other sulthydryl reagents do not have a similar effect is unclear, but dithiothreitol prevents the effects of thimerosal on CICR. 47. •
CARROLLJ, SWANN K: Spontaneous Cytosolic Calcium OsciUations Driven by Inositol Trisphosphate O c c u r during in Vitro Maturation of Mouse Oocytes. J Biol Chem 1992, 267:11196-11201. Heparin injection blocks lnsPyinduced Ca 2 + oscillations, but does not affect thimerosal actions on CICR. 48.
WHAILEYT, MCDOtlGAU. A, CRO.'~SLEY 1, SWANN K, WHITAKER M: Internal Calcium Release and Activation of Sea Urchin Eggs by cGMP are I n d e p e n d e n t of the Phosphoinositide Signaling Pathway. Mol Biol Cell 1992, 3:373-383. cGMP causes delayed Ca 2+ release in intact eggs, but not in digitoninpermeabilized eggs. The action of cGMP is insensitive to heparin, which suggests that cGMP activates an InsPyindependent Ca 2+-release pathway. In eggs previously activated by a poorly metabolized lnsP 3 analog, neither cGMP nor A23187 cause Ca 2+ release. This result suggests a single Ca 2+ store in the egg. •
49.
DARGIEPJ, AGILE MC, LEE HC: Comparison of Ca 2+ Mobilizing Activities of Cyclic-ADP Ribose and Inositol Trisphosphate. Cell Regul 1990, 1:279-290.
50.
RUSINKON, LEE HC: Widespread O c c u r r e n c e in Animal Tissues of an Enzyme Catalyzing the Conversion of NAD + into a Cyclic Metabolite with Intracellular Ca2+-Mobilizing Activity. J Biol Cbem 1989, 264:11725-11731.
51.
KOSHIYAMAH, LEE HC, T&SHJIAN AH: Novel Mechanism of lntracellular Calcium Release in Pituitary Cells. J Biol Cbem 1991, 266:16985-16988.
52.
IW,V~IAI,'SUT, YOSHIMOTOY, HIRAMOTOY: Mechanism of Ca 2+ Release in M e d a k a Eggs Microinjected with Inositol 1,4,5Trisphosphate and Ca 2+. Dev Biol 1988, 129:191-197.
53.
DASCALN, LANDAUEM: Cyclic GMP Mimics the Muscarinic Response in X e n o p u s Oocytes: Identity of Ionic Mechanisms. Proc Natl Acad Sci U S A 1982, 79:3052-3056.
54.
WALTON PD, AIREY JA, Su'rKo JL, BECK CF, MIGNERY FA, S0DHOF TC, DEEP,NICK TJ, ELLISMANMH: Ryanodine and Inositol Trisphosphate Receptors Coexist in Avian Cerebellar Purkinje Neurons. J Cell Biol 1991, 113:1145-1157.
55.
MCPHERSONSM, MCPHEP~SON PS, MATHEWS L, CAbIPBELL KP, LONGOFJ: Cortical Localization of a Calcium Release Channel in Sea Urchin Eggs. J Cell Biol 1992, 116:1111-1121. In this study, ryanodine receptors are localized to the egg cortex by using polyclonal antibodies to striated muscle t3,anodine receptors. An ~380 kD protein is identified using immunoblot analysis. Immunoreactivity of file cortex is altered by treatment of corticies with caffeine, ryanodine, Ca 2+ or A23187. •
56. .
TERAS/,.KIM, HENSON J, BEGG D, KAMINERB, SARDET C: Characterization of Sea Urchin Egg Endoplasmic Reticulum in Cortical Preparations. Dev Biol 1991, 148:398--401. In this work, file cortical ER is stained by Dil and observed to colocalized with calsequestrin-like protein. The cortical ER often encircles cortical granules, and is thus apparently positioned for initiating cortical granule fusion and propagating the Ca 2+ wave. 57. •
TERASAKIM, SARDET C: Demonstration of Calcium Uptake and Release by Sea Urchin Egg Cortical Endoplasmic Reticulum. J Cell Biol 1991, 115:1031-1037. The ER of isolated Arbacia, but not other species of sea urchin, may be loaded with fluo-3. The cortical ER is shown to be a site of ATPdependent Ca 2 + sequestration and lnsPyinduced Ca 2 + release. 58. •
TERA~,AKIM, JAFFE LA: Organization of the Sea Urchin Egg Endoplasmic Reticulum and its Reorganization at Fertilization. J Cell Biol 1991, 114:929-940. The ER of intact eggs is stained with Dil and the application of confocal microscopy reveals an extensive cell-wide, interconnected comparmlent that is in continuous motion. Tile ER became reorganized coincident with the Ca 2 + wave at fertilization, but by 5--8 rain it had returned to an organization nomlally seen in unfertilized eggs. A possible role for ER reorganization and sperm pronuclear events is discussed. 59. .,
LEE HC: Specific Binding of Cyclic ADP-ribose to Calciumstoring Microsomes from Sea Urchin Eggs. J Biol Chem 1991, 266:2276--2281. Specific binding of cADPR to a saturatable site on the Ca 2+ -storing microsomes is reported. The binding is unaffected by NAD + , ADP-ribose, heparin and InsPy Tbe binding shows a pH optimum at about 6.7 and is Ca 2+ -sensitive, which differs from the binding of InsP3 to its receptor. 60.
ZUCKERRS, STEINHARDT RA, WINKLER MM: lntracellular Calcium and Mechanisms of Parthenogenetic Activation of Sea Urchin Eggs. De,, Biol 1978, 65:285-295.
61.
TH,vs'rRuI'O, CULLEN PJ, DROBAK BK, HANLEY MR, DAWSON AP: Thapsigargin, a T u m o r Promoter, Discharges Intracellular Ca 2+ Stores by Specific Inhibition of the Endoplasmic Reticulum Ca2+-ATPase. Proc Nail Acad Sci U S A 1990, 87:2466-2470.
62.
JAFFE LF: Sources of Calcium in Egg Activation: A Review. Det, Biol 1983, 99:256-276.
63. McCuU.OH DH, CHAMBERS EL: Fusion of Membranes during •• Fertilization. J Gen Physiol 1992, 99:137-175. Ea rly events of s p e m l - e g g interactions are ex:unined by capacitance and current m~k,;urements. These measurements suggest that egg activation requires sperm-egg fusion, which supports tile hYl)Othesis that egg activation is driven by a sperm cTtosolic factor. 64.
SW,~N K: A Cytosolic Sperm Factor Stimulates Repetitive Calcium Increases and Mimics Fertilization in Hamster Eggs. Det,elopment 1990, 110:1295-1302.
65.
DALE B, DEFEUCE LJ, EHRENSTEIN G: Injection of a Soluble Sperm Extract into Sea Urchin Eggs Triggers the Cortical Reaction. E.xperentia 1985, 41:1068-1070.
SS Shen, Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011, USA.
