Development 105, 343-349 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

343

Polyspermic eggs in the anuran Discoglossus pictus develop normally

RICCARDO TALEVI

Dipartimento di Biologia Evolutiva e Comparata, Universitd di Napoli, Via Mezzocannone 8, 80134 Napoli, Italy

Summary

Fertilization and development in 400 eggs of the anuranDiscoglossus pictus has been followed. In these eggssuccessful sperm interaction is restricted to a small areaof the animal dimple called DI and causes a rapiddepolarization. A high incidence of polyspermy (36 %)was detected by in vivo observations of fertilization coneformation. Polyspermic eggs gave rise to fertilizationpotentials comparable to monospermic eggs and devel-

oped normally. By using current-injection technique it isshown that sperm penetration is independent of mem-brane potential. The role of the egg envelopes in regulat-ing sperm-egg interaction is discussed.

Key words: polyspermy, anuran, membrane potential,Discoglossus pictus.

Introduction

Fertilization in anurans is monospermic. Sperm entry islimited to the animal hemisphere and causes a rapiddepolarization of the membrane potential (Maeno,1959; Ito, 1972; Cross & Elinson, 1980; Cross, 1981;Schlichter & Elinson, 1981; Iwao etal. 1981; Iwao, 1982;Gray et al. 1982; Charbonneau et al. 1983a; Jaffe et al.1983; Webb & Nuccitelli, 1985), which in some specieshas been suggested to be a block to polyspermy (Cross& Elinson, 1980; Grey et al. 1982; Charbonneau et al.1983a; Jaffe et al. 1983; Webb & Nuccitelli, 1985; Iwao,1987). The cortex of anuran eggs contains corticalgranules, which exocytose at fertilization forming aphysical and functional barrier against supernumeraryspermatozoa (Grey et al. 1976). On the contrary,urodele eggs are physiologically polyspermic, lack corti-cal granules (Hope et al. 1963; Picheral, 1977) andpenetration may occur at any point along the eggsurface causing minor changes in the membrane poten-tial (Charbonneau etal. 1983a; Iwao, 1985).

Discoglossus pictus, an anuran, has characteristicscommon to both orders. Fertilization occurs at arestricted area of the animal hemisphere, called theanimal dimple, generating a fertilization potential typi-cal of the Anura (Talevi et al. 1985), and recent studieshave shown that sperm gated ion channels are localizedin this area (Talevi et al. 1985; Nuccitelli et al. 1988;Talevi & Campanella, 1988). The cortical cytoplasm ofthe dimple has features similar to that of other anuraneggs. The remainder of the egg cortex is similar tourodeles, lacking cortical granules (Campanella, 1975).Finally, pricking the egg in regions outside the animaldimple lead to minor modifications of membrane poten-tial (Talevi et al. 1985), as observed during sperm

penetration of urodele eggs (Charbonneau et al. 1983a;Iwao et al. 1985). However, in D. pictus, such astimulation generates a cortical wave that reaches thedimple area where it triggers a typical activation poten-tial (Talevi etal. 1985).

The spermatozoa of Discoglossus pictus are particu-larly long, measuring 2-33mm. The spermatozoa arereleased, after hormonal stimulation, into the seminalvesicles (Mann et al. 1963; N'Diaye et al. 191 A) wherethey become organized in bundles. Such bundles aremotionless in the seminal liquid, but they gain motilityfor 14 s when in contact with uterine eggs or 1/10Ringer.

The present study addresses the description and thephysiology of polyspermic eggs in Discoglossus pictusand, in particular, the role played by the fertilizationpotential (FP).

Materials and methods

Adult Discoglossus pictus were captured in the neighbour-hood of Palermo, during February and September, and keptin aquaria at room temperature.

Gametes were checked to verify their normal morphologyand physiology. Batches of eggs that showed variabilities indimension, pigmentation or in the distribution of jelly werediscarded. Spermatozoa viability was verified by checking themotility upon dilution in 1/10 Ringer solution. The percent-age of normal fertilization was then determined. The eggswere put in a Petri dish and covered with 1/10 Ringer solutioncontaining (mil): NaCl 111, KC1 0-2, CaCl2 013, MgSO40-08, Hepes 2-5, final pH7-8. A drop of semen was added onthe eggs, simulating the normal mating (Heron-Royer, 1983).It is worth mentioning that sperm concentration does notappear to affect fertilization and development (see also

344 R. Talevi

Campanella et al. 1988). Successful sperm-egg interactionwas indicated by the regression of the animal concavity about20min after insemination. All inseminations were made withthe same procedure.

In vivo observationsEggs fertilized in 1/10 Ringer containing (HIM): NaCl 11-1,CaCl2 013, KC1 0-2, MgSO4 0-08, and Hepes 2-5, finalpH7-8, were observed 20min after fertilization. At this time,the number of fertilization cones present in the 'Dl' area wasdetermined (see also Talevi & Campanella, 1988). Polysper-mic eggs were separated from monospermic eggs and stored ina Petri dish containing 1/10 Ringer at room temperature. Theembryos were followed for normal development up to tadpoleformation.

In order to follow cone formation in vivo, eggs were placedon a microslide covered by a coverslip with a centrally locatedhole. Photographs were made using a Leitz Orthomat Micro-scope and Ilford Pan F Film.

Electron microscopyEggs were fixed at 45min after fertilization, with 2-5% V/Vglutaraldehyde in 0-2M-phosphate buffer, postfixed in2 % OsO4 W/V in phosphate buffer, dehydrated in increasingethanol and embedded in Epon 812. Serial semi-thin sectionswere made to check the presence of the last portion of spermnucleus in the fertilization cones. The sections were madewith an LKB ultramicrotome and stained with methyleneblue. For scanning electron microscopy, eggs fixed in a similarmanner were dehydrated in increasing ethanol and Freon 113.The samples were dried by the critical-point method, coatedwith gold and examined with a Cambridge scanning electronmicroscope.

Electrical recordingEggs were placed on a plastic Petri dish containing 1/10Ringer at room temperature. Microelectrodes (10-30 MQ)filled with 3M-KC1 or l-25M-potassium citrate were used forintracellular recording. Signals were amplified with an intra-cellular amplifier (WPI), recorded on an oscilloscope andstored either on paper (Gould, Ohio) or on FM tape.

In experiments where the membrane potential (RP) wasdepolarized by current injection, a differential amplifier wasused. The current was injected using an electrode of 1-5 MQ.resistance filled with potassium citrate connected with a Grassstimulator. An Agar bridge was used for ground.

trusions and there is no development (Talevi & Campa-nella, 1988). According to the number of fertilizationcones present, it is possible to distinguish polyspermicand monospermic eggs. Table 1 shows data from 358embryos (18 females). The percentage of monospermiceggs was 64 % (n = 230) and of polyspermic eggs 36 %(n = 128).

There was much variability in the percentage ofpolyspermy from different females. Embryos wereobserved after 2 days to check the percentage of normaldevelopment, which was 76% for monospermic eggsand 64% for polyspermic eggs. In the latter case,abnormal development of eggs with more than fivefertilization cones {n = 15) significantly lowers this per-centage. In fact, eggs with less than five cones showsimilar development to monospermic eggs (n = 113).

Fig. 1 shows a top view of the Dl area of a polysper-mic egg in which it is possible to observe two fertiliz-ation cones. The cones, which are similar to each other,vary only in size (40-70 ̂ m), and indicate that twospermatozoa have penetrated. Sperm penetration does

Results

Eggs in 1/10 Ringer solution were observed using adissection microscope, about 20min after fertilization.At this stage the dimple everts contemporaneously withthe dissolution of the jelly plug (Campanella, 1975) andit is possible to observe the surface of the dimple area.It was previously shown that this area is highly differen-tiated morphologically (Campanella et al. 1988) andphysiologically (Talevi & Campanella, 1988), and thenormal interaction and sperm penetration occurredonly in the central part of this area, called Dl area. Oneof the features of this area is the presence of fertilizationcones, which are morphological evidence for normalsperm penetration. On the other hand, sperm interac-tion with the plasma membrane outside the Dl areadoes not lead to fertilization cones but only to pro-

S

4

B

Fig. 1. Scanning electron micrographs of multiplefertilization cones. (A) A 'double' fertilization cone due tothe interaction of 2 sperm in close proximity. The arrowsshow the site of sperm penetration. x800. (B) Two distinctfertilization cones in the Dl area, pb, 1st polar body. x300.

Polyspermy in Discoglossus pictus egg 345

Table 1. Percentage of monospermic and polyspermiceggs

Clutch

123456789

101112131415161718

Percentage %

Number ofmono-spermic

eggs

7• r .

1111226

1321904

20121415—11

64

Number ofnormal

development

2

—W223

1121762

11101111-11

76

Number ofpoly-

spermiceggs

13i

m11

71

—123

18119

16341

36

Number ofnormal

development

8;4

5_—51

-•I-J

1110%.:I—-1

64

not occur simultaneously in all cones (Fig. 2), but with atime difference ranging from 1 to 3min. Such staggerremains constant during the process of sperm pen-etration and reabsorption of the cone.

To be sure that the presence of multiple fertilizationcones in Dl area is the sign of multiple penetration ofspermatozoa, serial semi-thin sections of polyspermiceggs were made (n = 10). Fig. 3A shows a light micro-scope picture of a polyspermic egg fixed at 45 min fromfertilization. Serial semi-thin sections were obtainedfrom this egg and Fig. 3B and C shows cross-sections ofthe fertilization cones found in the Dl area. In bothsections, it is possible to see that the sperm interactionwith Dl surface caused two typical fertilization cones inwhich sperm penetration is almost complete. The latterpart of the sperm has not yet penetrated the fertilizationcone shown in Fig. 3B while, in the second fertilizationcone, penetration is more advanced (Fig. 3C), similarto the sequence shown in vivo in Fig. 2.

In order to ascertain whether the different percent-age of polyspermy found in some batches was due tocharacteristics of the batch of spermatozoa selected,eggs from one animal were divided into groups andfertilized under constant conditions with sperm fromthree or four different males. As can be seen in Table 2,polyspermy does not depend on the sperm batch used

Fig. 2. Sequence of the late stages of sperm penetration in a polyspermic egg. (A,B) 40-43 min after fertilization, onespermatozoon completes penetration into a fertilization cone (arrow), pb, 1st polar body. X100. (C) High magnification of Bto show the final stages of sperm penetration (arrow). At this time the fertilization cone is very close to the fertilizationmembrane (fin). X460. (D) 44 minutes after fertilization a second spermatozoon starts penetrating into a second FC (arrow),that becomes closer to the fertilization membrane during the disappearance of the first FC. xlOO.

346 R. Talevi

Fig. 3. Correlation between number of fertilization cones and penetrating sperm. (A) Two distinct fertilization cones in Dlarea. x200. (B,C) Semi-thin section of the same egg shown in A. X1800. In both sections it is possible to see the spermnuclei (arrow) inside the fertilization cones. In B the last portion of spermatozoon is not yet incorporated (small arrows).

Table 2. Incidence of polyspermy in eggs inseminatedusing sperm from four different males

Clutch A

1 M(5)P(0)2 M(4)P(5)3 M(8)P(0)4 M(4)P(4)5 M(13)P(0)

M, monospermic; P,

Males

B

M(10)P(0)M(7)P(3)M(3)P(0)M(3)P(4)M(12)P(0)

polyspermic.

C

M(6)P(0)M(3)P(4)M(7)P(1)M(5)P(3)

D

M(1)P(4)M(3)P(0)

and therefore depends upon characteristics of the eggs.Since not all egg clutches display polyspermy, theoccurrence of this condition should be correlated withcharacteristics of some egg clutches.

Electrical measurementsTable 3 shows data from 20 eggs from 8 animals,

fertilized in 1/10 normal Ringer and displaying poly-spermy, with a mean of 2-5 fertilization cones per egg.As shown in the table, the values of the RP and FP(-17-0 ±0-6 mV and +18-1 ± 4-2 mV, respectively) aresimilar to those found in monospermic eggs(-17-8 ± 2-8 mV and +19-5 ± 6-8 mV) and 80% ofthese eggs developed normally.

In several experiments, the membrane potential wasdepolarized using a differential amplifier and a secondcurrent-injecting electrode (Fig. 4). After 30s from thebeginning of artificial depolarization sperm were added.Current injection and the resulting membrane depolar-ization alone do not activate eggs. Sixteen eggs fromfive different animals were studied (see also Table 4)with a mean RP of -18-7 ± 2-4 mV. Current injectionsof 50 to 300 nA depolarized the potential to+25 ± 5 mV. Ten eggs gave rise to a potential variationof 2-5 mV at 20-120 s after sperm addition, followed bya series of small oscillations of the membrane potential.Once current injection was stopped, the membrane

Polyspermy in Discoglossus pictus egg 347

Table 3. Transmembrane voltage measurements inpolyspermic eggs

Experiment

Restingpotential

(mV)

Fertilizationpotential

(mV)

Number offertilization

cones

1234567891011121314151617181920

Mean ±S.D.

-14-10-18-15-18-18-20-18-16-18-17-20-19-18-18-18-19-18-13-16

-17-05 + 0-6

+ 15+20+ 18+ 19+ 16+20+ 12+ 16+23+20+21+20+ 18+20+ 17+ 16+ 14+ 18+22+ 15

+ 18 + 4-2

23352224322322322223

2-5 ±0-8

potential assumed a value typical of the FP, while theoscillation continued with the same intensity and fre-quency as in untreated eggs. Nine of these eggs weremonospermic; however, only one developed into anormal larva, the others stopped at various stages ofdevelopment (Table 4). This could be ascribed to thedamage caused by the insertion of a second electrodeinto the egg, which, since they were utilized to injectcurrent, had very low resistance and large tips, causinga considerable loss of cytoplasm after removal. Theremaining six eggs behaved differently. Two of them,although fertilized, showed no change in the membranepotential after the current injection ceased, remainingat the same RP value as that recorded before injection(see experiments 1 and 2 in Table 4). Two other eggsdid not activate until the current injection had beendiscontinued and the potential had resumed its originalresting value. Experiments 7 and 12 in Table 4 showedno response at all. All experiments using currentinjection are summarized in Table 4.

Discussion

Polyspermy in Discoglossus pictus (Anura) eggs hasbeen studied and the following new findings haveemerged, (a) In comparison with other anuran amphib-ians, Discoglossus pictus eggs show a high incidence ofpolyspermy; (b) polyspermic eggs with less than fivesperm develop normally; (c) susceptibility to poly-spermy is a characteristic of the egg and does notdepend on sperm; (d) polyspermic eggs have a normalfertilization potential; (e) sperm penetration is notregulated by the depolarization of the plasma mem-brane.

Fertilization in anurans is typically monospermic.

L

I-

-»ff t -

, - * '

L~—

f

5>»

A

"1 1

LFig. 4. Schematic drawing of experiments where themembrane potential was depolarized by current injection.V = electrodes connected to the differential amplifiermonitoring the transmembrane potential. I = current-injection electrode. The top trace shows the response of theegg potential to current injection (bottom trace).A • = start and end of current injection. A = sperm added,f = fertilization potential. Scale, Top trace, verticalbar = 10mV, horizontal bar = 10 s. Bottom trace, verticalbar = 100 mV, horizontal bar = 10 s.

The rate of polyspermy is very low and is incompatiblewith development. Elinson (1975) found only 4 poly-spermic eggs from a total of 1200 Rana pipiens eggs.Picheral & Charbonneau (1982) reported from 500 eggsof Rana temporaria, Rana pipiens and Rana esculenta a0-4% polyspermy rate. On the contrary, in vivo obser-vations of Discoglossus pictus eggs have shown that ahigh percentage of polyspermy (33-5 %) is compatiblewith normal development. Recently Elinson (1987)found a low rate of polyspermy compatible with thenormal development in the egg of the anuran Eleu-therodactylus coqui.

Polyspermy in the present report on Discoglossuspictus eggs is not due to immaturity, since immatureeggs do not generate normal FPs (Talevi et al. 1985).The polyspermic eggs studied here generated normalFPs and developed into normal tadpoles. Since polys-permic egg batches were always polyspermic when

348 R. Talevi

Table 4. Current-induced depolarization in D. Pictus eggs

Exp. RP* APt MP§ FDD FA|| Development

12345678910111213141516

-12-12-18-6-18-20-18-18-10-18-20-20-19-20-181 C

+25+30+30+20+20+20+30+25+25+20+25+20+30+25+30+25

1108070301001001206012012010030060809050

-12-12+25+6+18+18-18+21-10+18+20-20+20+ 19+ 18+ 18

YesYesYes—YesYes_Yes-YesYes_YesYesYesYes

* Resting potential (mV).t Artificial potential (mV) due to current injection.t Injection of current (nA).§ Membrane potential just after current turning off.H Fertilization during current injection.|| Fertilization after current turning off.

Yes

Yes

Early cleavageEarly cleavageBlastulaEarly cleavageGastrulaLarva

Blastula

Mid cleavageGastrulation

Mid cleavageEarly cleavageNeurulaBlastula

inseminated with different sperm batches, this does notdepend on characteristics of the sperm but on intrinsicfeatures of particular egg clutches.

Studies in progress on karyology show that poly-ploidy in Discoglossus pictus is not atypical. In nature,many triploid larvae have been found (Morescalchi,personal communication), suggesting the possibility ofreorganization of chromosome pattern. However, poly-ploidy has been found in other frogs such as Rananigromaculata and Rana brevipoda (Okumoto, 1983).

In a previous paper (Talevi et al. 1985), Discoglossuspictus eggs at fertilization have been shown to generatea change in resting potential typical of anuran Am-phibia (Maeno, 1959; Ito, 1972; Cross & Elinson, 1980;Grey et al. 1982; Schlichter & Elinson, 1981; Charbon-neau etal. 1983a,b; Iwao etal. 1981; Webb & Nuccitelli,1985).

It has been often been suggested (Cross & Elinson,1980; Grey et al. 1982; Charbonneau et al. 1983a; Jaffeet al. 1983; Webb & Nuccitelli, 1985) that the fastdepolarization at fertilization in anuran eggs representsa fast block to polyspermy, similar to that reported forEchinodermata (Jaffe, 1976). The existence of a fastblock is supported by many (see reviews by Whitaker &Steinhardt, 1982; Jaffe & Gould, 1985; Nuccitelli &Grey, 1984), but refuted by others (Dale & Monroy,1981; Dale, 1987). Furthermore, recently it has beenshown in the Echinoderm egg of Lytechinus variegatusthat opening of ion channels at fertilization precedesfusion of the sperm and egg plasma membrane (Hinkleyet al. 1986; Longo et al. 1986). In Discoglossus pictus,polyspermic eggs show the same shift in voltage asmonospermic eggs. In addition, experimental manipu-lation of the membrane potential before and duringinsemination has shown that interaction and pen-etration of the sperm are not related to membranepotential. In some cases, however, current injection

may modify the membrane, disturbing the interactionbetween sperm and egg. Probably for this reason, intwo of the cases reported here, fertilization only oc-curred when the current injection had ceased.

The question arises, what does regulate sperm entry?One possibility is the conformation and limited area ofthe animal dimple. In fact, fertilization cone formationand normal penetration can only occur in a restrictedarea of the dimple bottom called Dl, with a diameter ofabout 200 ,um (Talevi & Campanella, 1988). The fertiliz-ation cone has a diameter ranging from 40 to 70 [im and,therefore, the Dl could accommodate about 5-8 cones.In vivo observations have shown that, in polyspermiceggs, the number of fertilization cones is generally 2-3.Only in one case were 8 fertilization cones observed. Afurther limitation to the number of sperm that caninteract with this area is probably the particular charac-teristics of the plasma membrane, the animal plug andthe other jelly layers surrounding the egg. In a previouspaper (Talevi & Campanella, 1988), it has been shownthat the animal plug plays an important role in directingthe sperm towards the Dl area. Since the animal plug ismade of fibrillar material (Ghiara, 1960) one couldhypothesize that the organization of such material givesrise to a series of set routes, through which only alimited number of sperm can progress to the vitellinecoat. In polyspermic batches of eggs, this structure maybe altered, facilitating multiple sperm entry.

The idea of limited areas for sperm entry is not new(Runnstrom, 1961), and recently attention has beenpaid to the possibility of extracellular mechanisms thatregulate sperm entry (Dale, 1985, 1987).

The functional regionalization of the Discoglossuspictus egg, together with the observation that spermentry in this species is independent of membranepotential, provides new evidence for alternative mech-anisms of sperm-egg interaction.

Polyspermy in Discoglossus pictus egg 349

I am grateful to Profs G. Ghiara, C. Campanella and B.Dale for suggestions and comments and to Mrs G. Falconeand G. Argenzio for photographic work. Supported by PhDprogram fund 'Biologia Evolutiva e del Differenziamento'and to a CNR project on the Biology of Fertilization to G.Ghiara.

References

CAMPANELLA, C. (1975). The site of spermatozoon entrance in theunfertilized egg of Discoglossus pictus (Anura): An electronmicroscopy study. Biol. Reprod. 12, 439-447.

CAMPANELLA, C , TALEVI, R., KLINE, D. & NUCCITELLI, R. (1988).The cortical reaction in the eggs of Discoglossus pictus: A studyof the changes in the endoplasmic reticulum at activation. DeviBiol. (in press).

CHARBONNEAU, M., MOREAU, M., PICHERAL, B. & GUERRIER, P.(1983a). Fertilization of amphibian eggs: A comparison ofelectrical responses between anurans and urodeles. Devi Biol.98, 304-318.

CHARBONNEAU, M., MOREAU, M., PICHERAL, B., GUERRIER, P. &VILAIN, J. P. (19836). Voltage noise changes duringmonospermic and polyspermic fertilization of mature egg of theanuran Rana temporaria. Dev. Growth Differ. 25, 485-494.

CROSS, N. L. (1981). Initiation of the activation potential by anincrease in intracellular calcium in eggs of the frog Rana pipiens.Devi Biol. 85, 380-385.

CROSS, N. L. & ELINSON, R. P. (1980). A fast block to polyspermyin frogs mediated by changes in the membrane potential. DeviBiol. 75, 187-198.

DALE, B. (1985). Sperm receptivity in sea urchin oocytes and eggs.J. exp. Biol. 118, 85-97.

DALE, B. (1987). Mechanism of fertilization. Nature, Lond. 325,762-763.

DALE, B. & MONROY, A. (1981). How is polyspermy prevented?Gamete Res. 4, 151-169.

ELINSON, R. P. (1975). Site of sperm entry and a corticalcontraction associated with egg activation in the frog Ranapipiens. Devi Biol. 47, 257-268.

ELINSON, R. P. (1986). Fertilization in amphibians: the ancestry ofthe block to polyspermy. Int. Rev. Cyt. 101, 59-100.

ELINSON, R. P. (1987). Fertilization and aqueous development ofthe Puerto Rican terrestrial-breeding frog, Eleutherodactyluscoqui. J. Morph. 193, 217-224.

GHIARA, G. (1960). Ricerche intorno alia struttura microscopica,submicroscopica ed istochimica e alle funzioni degli involucriovulari di Discoglossus pictus Otth e di altre speccie di anfibi.Archo Zool. hal. 45, 9-92.

GREY, R. D., WORKING, P. K. & HEDRICK, J. L. (1976). Evidencethat the fertilization envelope blocks sperm entry in eggs ofXenops laevis: Interaction of sperm with isolated envelopes. DeviBiol. 54, 52-60.

GREY, R. D., BASTIANI, M. S., WENN, D. J. & SCHERTEL, E. R.(1982). An electrical block is required to prevent polyspermy ineggs fertilized by nature mating of Xenopus laevis. Devi Biol. 89,475-484.

HERON-ROYER, M. (1983). Le Discoglosse du Nord de l'Afrique(Discoglossus auritus H.R.) et son acclimation en France. Rev.Sci. Nat. appl. 38, 509.

HINKLEY, R. E., WRIGHT, B. D. & LYNN, J. W. (1986). Rapidvisual detection of sperm-egg fusion using the DNA-specificfluorochrome Hoechst 33342. Devi Biol. 118, 148-154.

HOPE, J., HUMPHRIES, A. A., JR & BOURNE, G. M. (1963).Ultrastructural studies on developing oocytes of the salamanderTriturus viridescens. J. Ultrastr. Res. 9, 302-324.

ITO, S. (1972). Effects of media of different ionic composition onthe activation potential of anuran egg cells. Dev. Growth Differ.14, 217-227.

IWAO, Y. (1982). Differential emergence of cortical granulebreakdown and electrophysiological responses during meioticmaturation of Toad oocytes. Dev. Growth Differ. 24, 467-477.

IWAO, Y. (1985). The membrane potential changes of amphibianeggs during species and cross-fertilization. Devi Biol. I l l , 26-34.

IWAO, Y. (1987). The spike component of the fertilization potentialin the toad, Bufo japonicus: changes during meiotic maturationand absence during cross-fertilization. Devi Biol. 123, 559-565.

IWAO, Y., ITO, S. & KATAGIRI, C. (1981). Electrical properties ofToad oocytes during maturation and activation. Dev. GrowthDiffer. 23, 89-100.

IWAO, Y., YAMASAKI, H. & KATAGIRI, C. (1985). Experimentspertaining to the suppression of accessory sperm in fertilizedNewt eggs. Dev. Growth Differ. 27, 323-331.

JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs iselectrically mediated. Nature, Lond. 261, 68-71.

JAFFE, L. A., CROSS, N. L. & PICHERAL, B. (1983). Studies of thevoltage-dependent polyspermy block using cross-speciesfertilization of amphibians. Devi Biol. 96, 317-323.

JAFFE, L. A. & GOULD, M. (1985). Polyspermy-preventingmechanisms. In Biology of Fertilization (ed. A. Monroy and C.B. Mertz). New York: Accademic Press.

LONGO, F. J., LYNN, J. W., MCCULLOH, D. H. & CHAMBERS, E. L.(1986). Correlative ultrastructural and electrophysiologicalstudies of sperm-egg interaction of the sea urchin, Lytechinusvariegatus. Devi Biol. 118, 155-166.

MAENO, T. (1959). Electrical characteristics and activation potentialof Bufo eggs. /. gen. Physiol. 43, 139-157.

MANN, T., LUTWAK-MANN, C. & HAY, M. F. (1963). A note on theso-called seminal vesicles of the frog Discoglossus pictus. ActaEmbryol. Morph. exp. 6, 21.

N'DIAYE, A., SANDOZ, D., BOLSIEVIEUX-ULRICH, E. & OZON, R.(1974). Action des androgenes chez l'Amphibien AnoureDiscoglossus pictus. IV. Etude ultrastructurale des phenomenesde secretion dans les vesicules seminales pendant la periodl'accouplement. J. Microbiol. 21, 309.

NUCCITELLI, R. & GREY, R. D. (1984). Controversy over the fast,partial, temporary block to polyspermy in sea urchin: areevaluation. Devi Biol. 103, 1-17.

NUCCITELLI, R., KLINE, D., BUSA, W., TALEVI, R. & CAMPANELLA,C. (1988). The activation current and intracellular calciumincrease in the egg of the frog Discoglossus pictus. Devi Biol. (inpress).

OKUMOTO, H. (1983). Studies on meioses in male hybrids andtriploids in the Rana nigromaculata group. II. Auto andAllotriploids in Rana nigromaculata and Rana brevipoda. Sci.Rep. Lab. Amphibian Biol., Hiroshima Univ. 6, 183-205.

PICHERAL, B. (1977). La fecondation chez le triton pleurodele.II. La penetration des spermatozoides et la reaction locale del'oeuf. J. Ultrastruct. Res. 60, 181-202.

PICHERAL, B. & CHARBONNEAU, M. (1982). Anuran fertilization: Amorphological reinvestigation of some early events. J. Ultrastr.Res. 81, 306-321.

RUNNSTROM, J. (1961). The mechanism of protection of the eggsagainst polyspermy. Experiments on the sea urchin Paracentrotuslividus. Ark. Zool. 13, 565-571.

SCHLICHTER, L. & ELINSON, R. P. (1981). Electrical responses ofimmature and mature Rana pipiens oocytes to sperm and otheractivating stimuli. Devi Biol. 83, 33-41.

TALEVI, R., DALE, B. & CAMPANELLA, C. (1985). Fertilization andactivation potentials in Discoglossus pictus (Anura) eggs: Adelayed response to activation by pricking. Devi Biol. Ill ,316-323.

TALEVI, R. & CAMPANELLA, C. (1988). Fertilization in Discoglossuspictus (Anura) I: Sperm-egg interactions in distinct regions ofthe dimple and occurrence of a late stage of sperm penetration.Devi Biol. (in press).

WEBB, D. J. & NUCCITELLI, R. (1985). Fertilization potential andelectrical properties of the Xenopus laevis egg. Devi Biol. 107,395-406.

WHITAKER, M. S. & STEINHARDT, R. A. (1982). Ionic regulation ofegg activation. Q. Rev. Biophys. 15, 593-666.

{Accepted 2 November 1988)