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Home , Cell fusion, Egg cell, Fertilisation, Gamete

Sex Reprod (2001) 14:21Ð26 © Springer-Verlag 2001

REVIEW

A.F. Antoine á C. Dumas á J.-E. Faure · J.A. Feijó M. Rougier activation in flowering

Received: 2 December 2000 / Accepted: 7 June 2001

Abstract Compared to and , egg activa- cal event is the which involves exocyto- tion in flowering plants is still poorly understood be- sis of cortical granules and elevation of the fertilization cause of the inaccessibility and complexity of the fertili- envelope (Moser 1939). In Lychetinus pictus , a set zation process which is double and internal. However, of events are observed that begins with (see also Stricker the development of in vitro fertilization (IVF) systems in 1999; Whitaker and Steinhardt 1982) (1) immediate cor- maize and a few other plants, despite some limitations, tical [Ca2+] increase (the cortical flash) and membrane offers new possibilities for the study of early post- potential variations (the fertilization potential). Within fusional events and signals leading to egg activation un- the first 5 min, this is followed by (2) a global cytosolic der defined conditions. This review reports recent data [Ca2+] increase (the Ca2+ wave), cortical reaction, activa- on events induced by fusion during tion of kinases and phosphates, proton efflux and in- maize IVF and presents perspectives on the role of calci- crease in O2 consumption (the respiration burst). Within um in egg activation and in early development. 15Ð20 min we observe (3) initiation of traduction then of replication. Within 60Ð80 min there is (4) and Keywords Zea mays á In vitro fertilization á Egg within 85Ð95 min (5) the first division. activation á Calcium Egg activation has been the subject of numerous in- vestigations in both animals and algae (Roberts and Brownlee 1995; Stricker 1999), and involvement of a cy- 2+ 2+ Introduction: tosolic [Ca ] (cyt[Ca ]) increase has been proposed as a egg activation or how to initiate a new ? key event leading to egg activation at an early stage (Jaffe 1980). This hypothesis has now been validated in a num- can be viewed as quiescent cells which ber of species. So far, every species studied shows at least 2+ require fertilization for reactivation. Fertilization triggers a cyt[Ca ] elevation after gamete fusion (for review see “egg activation”, a preprogrammed set of events that Ciapa and Chiri 2000; Stricker 1999). However, each leads to the development of a new individual. These system is characterized by a unique spatio-temporal pat- 2+ events fall into three broad categories: morphological, tern of the cyt[Ca ] elevation. For instance, in Lychetinus physiological and biochemical. Each event is precisely pictus eggs, gamete fusion induces an immediate Ca2+ in- timed and plays a unique role in the activation of devel- flux through voltage-activated calcium channels (the cor- 2+ opment. For instance, in sea urchins, where egg activa- tical flash) during which the cyt[Ca ] elevation is restrict- tion is well documented, the most obvious morphologi- ed to the cortical region of the egg (Shen and Buck 1993). 2+ Then, within 500Ð3000 s a global cyt[Ca ] increase A.F. Antoine (✉) á C. Dumas á J.E. Faure á M. Rougier spreads as a wave across the (Gillot and Whitaker Laboratoire de et Développement des plantes. 1993; Steinhardt et al. 1977). In contrast, in the echiurian UMR 5667 CNRS, INRA, UCB Lyon 1, ENS Lyon, Urechis caupo gamete fusion induces a single Ca2+ tran- 46 Allée d’Italie, 69364 Lyon Cedex 07, France e-mail: [email protected] sient that does not spread in a wavelike fashion (Stephano Tel.: +33-4-72-72-86-14, Fax: +33-4-72-72-86-00 and Gould 1997). A Ca2+ influx is necessary for this [Ca2+] elevation, although simultaneous Ca2+ release J.A. Feijó cyt Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal from internal stores may also be required. 2+ The proposition that a cyt[Ca ] elevation is necessary Present address: and sufficient for egg activation was also made early (for J.A. Feijó, Centro de Biotecnologia Vegetal, Faculdade de Ciências, Univ. de Lisboa, Campo Grande, review see Epel 1990; Steinhardt et al. 1974; Whitaker Ed.C2, 1749-016 Lisboa, Portugal and Swann 1993). To demonstrate this, it is necessary to 22

2+ prove that (1) an artificial cyt[Ca ] elevation triggers egg present only during a defined time period between polli- activation in unfertilized eggs and that (2) preventing the nation and the first zygotic division, and it may play a 2+ cyt[Ca ] elevation during fertilization inhibits egg acti- role in gamete adhesion and fusion. Polarization pre- vation. Such demonstrations have so far been attempted cedes the first zygotic division; in maize the mass of the only in few species, namely, , ascidians and which initially occupies a central position (see Evans and Kopf 1998; Stricker 1999). shifts to the apical pole of the (Mòl et al. 1994). In plants, egg activation was first analyzed in easily Historically, the development of in vitro fertilization accessible systems such as fucoid algae (Brownlee (IVF) systems has proved to be useful for analysis of egg 2+ 1994). In , egg activation is triggered by a cyt[Ca ] activation in animals and algae. In flowering plants, de- increase which is localized to the cortical region and velopment of such systems has been delayed due to the takes place simultaneously throughout the egg (Roberts necessity of isolating male and female gametes (for re- 2+ 2+ et al. 1994). This cyt[Ca ] increase results from a Ca view see Chaboud and Perez 1992; Theunis et al. 1991) influx through voltage-gated calcium channels that is before performing in vitro fusion (for review see Kranz triggered by fertilization (Taylor and Brownlee 1993). 1999; Kranz and Dresselhaus 1996). The earliest plant Minutes after fusion, exocytosis of the cortical granules IVF system was developed by Kranz and co-workers is observed and a new is synthesized (Brawley (Kranz et al. 1991) who used electric pulses to induce fu- et al. 1976a). Subsequently, the zygote starts to polarise sion between an and a male gamete in Zea mays. (Brawley et al. 1976b) and the first is un- Using this IVF system, whole plants could be regenerat- equal (for review see Robinson et al. 1999). Thus, mech- ed from in vitro fusion products using feeder cells anisms of egg activation in algae and in animals, which (Kranz and Lörz 1993). The same technique was also both use naturally free-living gametes for fertilization, used to produce in vitro by fusing a male ga- appear analogous. In flowering plants, the situation is mete and the central cell (Kranz et al. 1998). In maize, different and more complex because fertilization is dou- alternative IVF systems were also developed which used ble and internal. During , two male Ca2+ as a mediator of fusion instead of an electrical pulse gametes undergo gametic fusion; one fuses with the egg (Faure et al. 1994; Kranz and Lörz 1994). IVF systems cell, giving rise to the , and the other fuses with were also developed in other species (Coix, Sorghum, the central cell, giving rise to the endosperm (Russell Hordeum, Triticum and Nicotiana) using either electro-, 1992). In contrast to animals and algae, male gametes calcium- or PEG-mediated fusion (Kovacs et al. 1994; seem to be non-motile and female gametes are deeply Kranz et al. 1995: Sun et al. 2000). All of these IVF sys- embedded in the female tissues. Given these differences, tems give direct access to the egg activation events in- it is important to investigate whether egg activation in duced by gametic fusion, and to early embryogenesis flowering plants follows the same principle as in other (for review see Dumas and Faure 1995; Rougier et al. . 1996; Kranz 1999; Faure and Dumas 2001). Despite the usefulness of these IVF systems for the study of egg activation in flowering plants, data obtained What do we know about egg activation should always be carefully viewed in light of the fact in flowering plants? that in vitro fertilization conditions differ from those in vivo. In vivo, fertilization processes take place inside Events triggering egg activation following in vivo fertili- maternal tissues which can interact with the egg cell. In zation in flowering plants are poorly understood. A num- contrast, IVF systems permit analysis of fusion and acti- ber of ultrastructural studies in various species have re- vation events without the influence of maternal tissues ported reorganization of the egg cell following its fertili- and under defined conditions. Another difference is that zation (for reviews, see Russell 1993; Van Went and the male gametes used for IVF are isolated directly from Willemse 1984). Changes in zygote volume and in the mature instead of being liberated following their number of ribosomes or organelles such as endoplasmic transport through the pistil inside a , a step reticulum or dictyosomes have been reported in addition that may be important for their final maturation. In addi- to the synthesis of a cell wall around the zygote. In tion, egg cells used for IVF (with the exception of barley, maize, data is derived mainly from electron microscopy wheat and rape , see Kranz 1999) are treated with studies (Diboll 1968; Van Lammeren 1986). The most cell wall degrading , a treatment which may af- documented aspect of maize egg activation is the in- fect their physiology. Finally, the in vitro fusion media, volvement of the cytoskeleton. induces accu- used at present, contain no to support the meta- mulation of patches in the cytosol which later accu- bolic reactivation undergone during egg activation. This mulate in the cortical region (Huang et al. 1999). Both step is necessary for completion of the first zygotic divi- actin reorganization in the egg cell and similar actin mo- sion that can therefore only be obtained by co-culture bilization in the synergids lead to formation of the actin with feeder cells (Kranz and Lörz 1993). However, for corona. This actin corona is an important feature of dou- the study of the earliest steps of egg activation, the in ble fertilization, and is also observed in Plumbago vitro fertilization approach has proved to be very useful (Huang et al. 1993), Nicotiana (Huang and Russell 1994) (for review see Dumas and Faure 1995; Faure and and Torenia (Huang et al. 1999). The actin corona is Dumas 2001; Kranz 1999; Rougier et al. 1996). Howev- 23 er, since the kinetics of fertilization are imprecise (Mòl ability may also reflect differences in the physiology of et al. 1994), gamete fusion cannot be controlled and it is the interacting cells. Although we only use mature male therefore difficult to study the earliest postfusion events. gametes, their physiological state may vary since they The majority of data concerning egg activation in are isolated from a mixed population of pollen grains flowering plants is derived mainly from IVF studies per- which are produced continuously over 1Ð2 days by a sin- formed in maize. The cellular events of egg activation gle male . Similarly, the egg cells come from a tas- have been analyzed in this plant using both electrofusion sel that presents a gradient of flower maturity (see Kranz (Kranz et al. 1991) and calcium-mediated maize IVF 1999). However, one stricking feature emerges from performed at low pH (Faure et al. 1994). In both cases, these data: in every experiment performed so far, a 2+ is completed within 1 h after egg- fu- cyt[Ca ] elevation, as has been described in all other or- sion (Faure et al. 1993; M. Rougier, unpublished data), ganisms, is systematically triggered after gamete fusion indicating that the earliest steps of egg activation take (Digonnet et al. 1997; A. F. Antoine, unpublished re- place during this period. Fusion is frequently followed sults) . 2+ by a transient egg cell deformation and by organelle con- The hypothesis that the observed cyt[Ca ] elevation densation around the nucleus, which then takes a periph- plays a role in maize egg activation remains unproven. 2+ eral position (Kranz et al. 1995; Antoine et al. 2000). Si- However, it has been shown that an artificial cyt[Ca ] el- multaneously, the fusion product is enclosed by a newly evation (induced by Ca2+ ionophore treatment) induces synthesized cell wall (the egg cell is initially a true pro- some manifestations of egg activation (Antoine et al. toplast and, in the minimal fusion medium, does not syn- 2000). Taken together with the raised levels of the calci- thesize a cell wall until at least 48 h after fertilization). It um-calmodulin complex observed in in vitro should be stressed that contrary to the situation in algae, (Tirlapur et al. 1995), this result strongly suggests a role cell wall establishment does not initially result from a for Ca2+ in egg activation in flowering plants, possibly massive exocytosis of cortical vesicles. The maize egg via a calmodulin-mediated Ca2+ signaling pathway. 2+ cell does not contain any accumulation of cortical vesi- However, a demonstration that inhibition of the cyt[Ca ] cles (Faure et al. 1992). However, as in Fucus, establish- elevation prevents egg activation is still lacking. As in ment of a new extracellular matrix could well account other IVF systems, this could be achieved by treatment for an apparent block to observed minutes with cell-permeant Ca2+ buffers such as BAPTA-AM after the first successful fertilization. Indeed, for zygotes (McDougall et al. 1995; Stephano and Gould 1997; produced by calcium-mediated fusion (Faure et al. Lawrence et al. 1998; Raz et al. 1998). 1994), a second fusion has never been observed later than 45 s after the first fusion event, when cell wall syn- thesis is initiated (Kranz et al. 1995). The phenomena Which mechanisms 2+ described are the earliest egg activation events that have of cyt[Ca ] increase during maize IVF? been characterized in maize at the cellular level. Similar 2+ events appear to occur during IVF in other plant species Detection of cyt[Ca ] elevation after maize fertilization such as Coix, Sorghum, Hordeum, Triticum and Nicoti- leads inevitably to the question of its origin. In plants, ana (Kranz et al. 1995; Sun et al. 2000). calcium can be mobilized from the extracellular medium and/or from internal stores (Hamon 1997; McAinsh et al. 1997; Sanders et al. 1999). The first hypothesis has been Does Ca2+ play a role in maize egg activation? investigated using a calcium-selective vibrating probe, a non-invasive technique well adapted for measurement of 2+ A cyt[Ca ] elevation has been observed during maize ionic fluxes (for review see Kühtreiber and Jaffe 1990; IVF using Fluo-3 calcium-imaging (Digonnet et al. Smith et al. 1994; Shipley and Feijò 1999). Such a spa- 1997). This occurs as a single transient elevation trig- tio-temporal analysis was made possible only because of gered solely by gamete fusion and not by adhesion alone. the particularities of the IVF used (Faure et al. 1994), It shoud be noted that the abruptness and the duration of where position and timing of gamete fusion is deter- 2+ 2+ this cyt[Ca ] elevation is variable from one experiment mined by simple visual observation. Onset of a Ca in- 2+ to the next. The cyt[Ca ] rise may be instantaneous or flux into the egg cell has been detected immediately after may take up to 20 s to be established. The duration of the gametic fusion. In the vicinity of the sperm fusion site, 2+ cyt[Ca ] elevation may range from 10 min to more than this influx starts immediately at the beginning of sperm 1 h (A. F. Antoine, unpublished data). Variability in oth- incorporation (time resolution is around 1 s). Initiation of er parameters linked to egg activation has frequently a Ca2+ influx then spreads in a wave-like fashion to the been observed during maize IVF, for example the vari- whole plasma membrane with a propagation rate in the able intensity of egg cell contraction (Antoine et al. range of 1 µm secÐ1 (Antoine et al. 2000 and see Fig. 1). 2+ 2+ 2+ 2000; Kranz et al. 1995) or the amount of Ca entering The contribution of the Ca influx to cyt[Ca ] eleva- the egg cell during the Ca2+ influx (Antoine et al. 2000). tion is still unknown and three hypothesis should be con- Such variability is not surprising since the number of ex- sidered. (1) The Ca2+ influx could directly participate in 2+ periments performed is limited by the inherent difficul- the cyt[Ca ] increase as was described in Fucus, where 2+ 2+ ties of the IVF system (see Kranz 1999). However, vari- cyt[Ca ] elevation results entirely from the Ca influx 24

Fig. 1 Onset of a Ca2+ influx during maize in vitro fertilization. As soon as fusion takes place (A), a Ca2+ influx into the egg cell is triggered at the male gamete incorporation site (B). The Ca2+ in- flux subsequently spreads to the whole fusion product plasmalem- ma in a wave-like fashion with a velocity in the range of 1 µm.secÐ1 (C). When set, the Ca2+ influx lasts for minutes and has an homogenous intensity all over the plasmalemma (D) Fig. 2 Hypothetical chronology of the earliest events induced by fertilization in maize. Time zero is set at the moment of membrane (Roberts and Brownlee 1995). However, (2) it is possible fusion. Fusion is immediately followed by male gamete incorpora- 2+ tion into the egg cell. Fusion is also immediately followed by the that the Ca influx could contribute indirectly to the 2+ 2+ 2+ onset of a Ca influx that propagates in a wave-like fashion from cyt[Ca ] elevation by refilling Ca stores, as is the case the fusion site to the whole of the fusion product plasmalemma. in mammals (for recent reviews see Swann and Parring- 2+ From 10Ð60 s after fusion, a cyt[Ca ] increase is observed and the ton 1999; Wassarman 1999). Indeed, the Ca2+ influx into fusion product goes through a transient deformation. From 10Ð the maize egg cell may be buffered by Ca2+ pumping in- 60 min after fusion, the fusion product starts to polarize and a cell side the vacuoles. This hypothesis is supported by the wall is synthesized. Karygogamy takes place after about 1 h, giv- ing rise to the zygote itself observation that the vacuoles comprise more than 85% of the maize egg cell (Faure et al. 1992) and are located just beneath the plasma membrane. Lack of quantitative zygote polarization (for a general discussion see Jaffe imaging of [Ca2+] elevation has prevented verification cyt 1999; Cove 2000). Such a role during maize fertilization of this hypothesis (Digonnet et al. 1997). Finally, (3) it is is further suggested by a wealth of data now emerging, possible that Ca2+ influx may not be necessary for which shows that the spatio-temporal patterns of [Ca2+] [Ca2+] elevation. This hypothesis is based on investi- cyt cyt variations are closely linked to the establishment of polar- gations carried out in sea urchin eggs where a Ca2+ in- ity in plant cells (for recent reviews see Brownlee et al. flux (the cortical flash) is instantly triggered by gamete 1999; Malhò et al. 1998; Trewavas 1999). fusion (Paul and Johnston 1978). However, this cortical Finally, downstream events induced by the [Ca2+] flash is not necessary for subsequent [Ca2+] elevation cyt cyt elevation shoud not be overlooked since these events are (McDougall et al. 1993; Shen and Buck 1993) which re- involved in the earliest steps of embryo development. lies mostly on IP -mediated Ca2+ release (see Stricker 3 Here again, the IVF systems could prove a very useful 1999 for extended discussion). It would appear that the tool. Using the maize IVF system, levels of activity cortical flash may not be necessary for sea urchin egg ac- during fertilization are now being studied, thanks to the tivation either, although this question is still under de- development of cDNA libraries from maize gametes and bate (see Creton and Jaffe 1995; Jones et al. 1998). from in vitro zygotes (Dresselhaus et al. 1994). By dif- ferential screening of these libraries, that are pref- Future prospects erentially expressed in the zygote have already been iso- lated (Dresselhaus et al. 1996, Sauter et al. 1998). Some genes appear to be associated with and An up-to-date summary of the egg activation events in control of the . One of the major future chal- maize is proposed in Fig. 2. Mobilization of the intracel- lenges will be to unravel the cascade of events leading lular Ca2+ store has yet to be assessed and thus is not from gamete fusion to the development of a new multi- mentioned in the figure. However, all of the tools neces- 2+ cellular organism. sary for analysis of the mechanisms of cyt[Ca ] elevation are currently available. Much of our work is now aimed Acknowledgements Thanks are due to Gwyneth Ingram for care- at assessing the respective contributions of Ca2+ influx fully reading and correcting the manuscript. (This manuscript was and of Ca2+ mobilization from intracellular stores to the included in the special edition of the journal in honor of Joseph 2+ Mascarenhas.) cyt[Ca ] elevation. Furthermore, the spatio-temporal pat- 2+ tern of cyt[Ca ] elevation is now under study by confo- cal Ca2+ imaging. This analysis will not only complete the description of the egg activation events, but could References also help the understanding of the mechanisms responsi- Antoine AF, Faure J-E, Cordeiro S, Dumas C, Rougier M, Feijó ble for acquisition of polarity in the zygote. Numerous JA (2000) A calcium influx is triggered and propagates in the studies of fertilization have shown that the spa- zygote as a wavefront during in vitro fertilization of flowering 2+ tio-temporal pattern of cyt[Ca ] elevation is linked to plants. 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