Fertilization and development in 400 eggs of the anuran Discoglossus pictus has been followed. In these eggs successful sperm interaction is restricted to a small area of the animal dimple called DI and causes a rapid depolarization. A high incidence of polyspermy (36 %) was detected by in vivo observations of fertilization cone formation. Polyspermie eggs gave rise to fertilization potentials comparable to monospermic eggs and developed normally. By using current-injection technique it is shown that sperm penetration is independent of mem-brane potential. The role of the egg envelopes in regulating sperm–egg interaction is discussed.

Fertilization in anurans is monospermic. Sperm entry is limited to the animal hemisphere and causes a rapid depolarization of the membrane potential (Maeno, 1959; Ito, 1972; Cross & Elinson, 1980; Cross, 1981; Schlich ter & Elinson, 1981; Iwao et al. 1981; Iwao, 1982; Gray et al. 1982; Charbonneau et al. 1983a; Jaffe et al. 1983; Webb & Nuccitelli, 1985), which in some species has been suggested to be a block to polyspermy (Cross & Elinson, 1980; Grey et al. 1982; Charbonneau et al. 1983b; Jaffe et al. 1983; Webb & Nuccitelli, 1985; Iwao, 1987). The cortex of anuran eggs contains cortical granules, which exocytose at fertilization forming a physical and functional barrier against supernumerary spermatozoa (Grey et al. 1976). On the contrary, urodele eggs are physiologically polyspermie, lack cortical granules (Hope et al. 1963; Picheral, 1977) and penetration may occur at any point along the egg surface causing minor changes in the membrane potential (Charbonneau et al. 1983a; Iwao, 1985).

Discoglossus pictus, an anuran, has characteristics common to both orders. Fertilization occurs at a restricted area of the animal hemisphere, called the animal dimple, generating a fertilization potential typical of the Anura (Talevi et al. 1985), and recent studies have shown that sperm gated ion channels are localized in this area (Talevi et al. 1985; Nuccitelli et al. 1988; Talevi & Campanella, 1988). The cortical cytoplasm of the dimple has features similar to that of other anuran eggs. The remainder of the egg cortex is similar to urodeles, lacking cortical granules (Campanella, 1975). Finally, pricking the egg in regions outside the animal dimple lead to minor modifications of membrane potential (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 a stimulation generates a cortical wave that reaches the dimple area where it triggers a typical activation potential (Talevi eí al. 1985).

The spermatozoa of Discoglossus pictus are particularly long, measuring 2·33mm. The spermatozoa are released, after hormonal stimulation, into the seminal vesicles (Mann et al. 1963; N’Diaye et al. 1974) where they become organized in bundles. Such bundles are motionless in the seminal liquid, but they gain motility for 14 s when in contact with uterine eggs or 1/10 Ringer.

The present study addresses the description and the physiology of polyspermie eggs in Discoglossus pictus and, in particular, the role played by the fertilization potential (FP).

Adult Discoglossus pictus were captured in the neighbourhood of Palermo, during February and September, and kept in aquaria at room temperature.

Gametes were checked to verify their normal morphology and physiology. Batches of eggs that showed variabilities in dimension, pigmentation or in the distribution of jelly were discarded. Spermatozoa viability was verified by checking the motility upon dilution in 1/10 Ringer solution. The percentage of normal fertilization was then determined. The eggs were put in a Petri dish and covered with 1/10 Ringer solution containing (mM): NaCl 11·1, KC1 0·2, CaCl2 0·13, MgSO4 0·08, Hepes 2·5, final pH 7·8. A drop of semen was added on the eggs, simulating the normal mating (Heron-Royer, 1983). It is worth mentioning that sperm concentration does not appear to affect fertilization and development (see also Campanella et al. 1988). Successful sperm-egg interaction was indicated by the regression of the animal concavity about 20 min after insemination. All inseminations were made with the same procedure.

In vivo observations

Eggs fertilized in 1/10 Ringer containing )mM): NaCl ll·l, CaCl2 0·13, KC1 0·2, MgSO4 0·08, and Hepes 2·5, final ρH 7·8, were observed 20 min after fertilization. At this time, the number of fertilization cones present in the ‘DI’ area was determined (see also Talevi & Campanella, 1988). Polyspermie eggs were separated from monospermic eggs and stored in a Petri dish containing 1/10 Ringer at room temperature. The embryos were followed for normal development up to tadpole formation.

In order to follow cone formation in vivo, eggs were placed on a microslide covered by a coverslip with a centrally located hole. Photographs were made using a Leitz Orthomat Microscope and Ilford Pan F Film.

Electron microscopy

Eggs were fixed at 45 min after fertilization, with 2 · 5% V/V glutaraldehyde in 0 · 2m-phosphate buffer, postfixed in 2% OsO4 W/V in phosphate buffer, dehydrated in increasing ethanol and embedded in Epon 812. Serial semi-thin sections were made to check the presence of the last portion of sperm nucleus in the fertilization cones. The sections were made with an LKB ultramicrotome and stained with methylene blue. For scanning electron microscopy, eggs fixed in a similar manner were dehydrated in increasing ethanol and Freon 113. The samples were dried by the critical-point method, coated with gold and examined with a Cambridge scanning electron microscope.

Electrical recording

Eggs were placed on a plastic Petri dish containing 1/10 Ringer at room temperature. Microelectrodes (10 – 30 MΩ) filled with 3M-KCl or 1·25M-potassium citrate were used for intracellular recording. Signals were amplified with an intracellular amplifier (WPI), recorded on an oscilloscope and stored either on paper (Gould, Ohio) or on FM tape.

In experiments where the membrane potential (RP) was depolarized by current injection, a differential amplifier was used. The current was injected using an electrode of 1– 5 MΩ resistance filled with potassium citrate connected with a Grass stimulator. An Agar bridge was used for ground.

Eggs in 1/10 Ringer solution were observed using a dissection microscope, about 20min after fertilization. At this stage the dimple everts contemporaneously with the dissolution of the jelly plug (Campanella, 1975) and it is possible to observe the surface of the dimple area. It was previously shown that this area is highly differentiated morphologically (Campanella et al. 1988) and physiologically (Talevi & Campanella, 1988), and the normal interaction and sperm penetration occurred only in the central part of this area, called DI area. One of the features of this area is the presence Of fertilization cones, which are morphological evidence for normal sperm penetration. On the other hand, sperm interaction with the plasma membrane outside the DI area does not lead to fertilization cones but only to protrusions and there is no development (Talevi & Campanella, 1988). According to the number of fertilization cones present, it is possible to distinguish polyspermie and monospermic eggs. Table 1 shows data from 358 embryos (18 females). The percentage of monospermic eggs was 64 % (n = 230) and of polyspermie eggs 36 % (n = 128).

Table 1.

Percentage of monospermic and polyspermie eggs

Percentage of monospermic and polyspermie eggs
Percentage of monospermic and polyspermie eggs

There was much variability in the percentage of polyspermy from different females. Embryos were observed after 2 days to check the percentage of normal development, which was 76% for monospermic eggs and 64% for polyspermie eggs. In the latter case, abnormal development of eggs with more than five fertilization cones (n = 15) significantly lowers this percentage. In fact, eggs with less than five cones show similar development to monospermic eggs (n = 113).

Fig. 1 shows a top view of the DI area of a polyspermie egg in which it is possible to observe two fertilization cones. The cones, which are similar to each other, vary only in size (40 – 70 μm), and indicate that two spermatozoa have penetrated. Sperm penetration does not occur simultaneously in all cones (Fig. 2), but with a time difference ranging from 1 to 3 min. Such stagger remains constant during the process of sperm penetration and reabsorption of the cone.

Fig. 1.

Scanning electron micrographs of multiple fertilization cones. (A) A ‘double’ fertilization cone due to the interaction of 2 sperm in close proximity. The arrows show the site of sperm penetration. ×800. (B) Two distinct fertilization cones in the DI area, pb, 1st polar body. ×300.

Fig. 1.

Scanning electron micrographs of multiple fertilization cones. (A) A ‘double’ fertilization cone due to the interaction of 2 sperm in close proximity. The arrows show the site of sperm penetration. ×800. (B) Two distinct fertilization cones in the DI area, pb, 1st polar body. ×300.

Fig. 2.

Sequence of the late stages of sperm penetration in a polyspermie egg. (A,B) 40 – 43 min after fertilization, one spermatozoon completes penetration into a fertilization cone (arrow), pb, 1st polar body. ×100. (C) High magnification of B to show the final stages of sperm penetration (arrow). At this time the fertilization cone is very close to the fertilization membrane (fin). ×460. (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. ×100.

Fig. 2.

Sequence of the late stages of sperm penetration in a polyspermie egg. (A,B) 40 – 43 min after fertilization, one spermatozoon completes penetration into a fertilization cone (arrow), pb, 1st polar body. ×100. (C) High magnification of B to show the final stages of sperm penetration (arrow). At this time the fertilization cone is very close to the fertilization membrane (fin). ×460. (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. ×100.

To be sure that the presence of multiple fertilization cones in DI area is the sign of multiple penetration of spermatozoa, serial semi-thin sections of polyspermie eggs were made (n = 10). Fig. 3A shows a light microscope picture of a polyspermie egg fixed at 45 min from fertilization. Serial semi-thin sections were obtained from this egg and Fig. 3B and C shows cross-sections of the fertilization cones found in the DI area. In both sections, it is possible to see that the sperm interaction with DI surface caused two typical fertilization cones in which sperm penetration is almost complete. The latter part of the sperm has not yet penetrated the fertilization cone shown in Fig. 3B while, in the second fertilization cone, penetration is more advanced (Fig. 3C), similar to the sequence shown in vivo in Fig. 2.

Fig. 3.

Correlation between number of fertilization cones and penetrating sperm. (A) Two distinct fertilization cones in DI area. ×200. (B,C) Semi-thin section of the same egg shown in A. ×1800. In both sections it is possible to see the sperm nuclei (arrow) inside the fertilization cones. In B the last portion of spermatozoon is not yet incorporated (small arrows).

Fig. 3.

Correlation between number of fertilization cones and penetrating sperm. (A) Two distinct fertilization cones in DI area. ×200. (B,C) Semi-thin section of the same egg shown in A. ×1800. In both sections it is possible to see the sperm nuclei (arrow) inside the fertilization cones. In B the last portion of spermatozoon is not yet incorporated (small arrows).

In order to ascertain whether the different percentage of polyspermy found in some batches was due to characteristics of the batch of spermatozoa selected, eggs from one animal were divided into groups and fertilized under constant conditions with sperm from three or four different males. As can be seen in Table 2, polyspermy does not depend on the sperm batch used and therefore depends upon characteristics of the eggs. Since not all egg clutches display polyspermy, the occurrence of this condition should be correlated with characteristics of some egg clutches.

Table 2.

Incidence of polyspermy in eggs inseminated using sperm from four different males

Incidence of polyspermy in eggs inseminated using sperm from four different males
Incidence of polyspermy in eggs inseminated using sperm from four different males

Electrical measurements

Table 3 shows data from 20 eggs from 8 animals, fertilized in 1/10 normal Ringer and displaying polyspermy, 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) are similar to those found in monospermic eggs (– 17·8±2 · 8mV and +19 · 5 ± 6 · 8mV) and 80% of these eggs developed normally.

Table 3.

Transmembrane voltage measurements in polyspermie eggs

Transmembrane voltage measurements in polyspermie eggs
Transmembrane voltage measurements in polyspermie eggs

In several experiments, the membrane potential was depolarized using a differential amplifier and a second current-injecting electrode (Fig. 4). After 30 s from the beginning of artificial depolarization sperm were added. Current injection and the resulting membrane depolarization alone do not activate eggs. Sixteen eggs from five different animals were studied (see also Table 4) with a mean RP of —18 · 7 ±2 · 4mV. Current injections of 50 to 300 nA depolarized the potential to +25 ± 5 mV. Ten eggs gave rise to a potential variation of 2 – 5 mV at 20 – 120 s after sperm addition, followed by a series of small oscillations of the membrane potential. Once current injection was stopped, the membrane potential assumed a value typical of the FP, while the oscillation continued with the same intensity and frequency as in untreated eggs. Nine of these eggs were monospermic; however, only one developed into a normal larva, the others stopped at various stages of development (Table 4). This could be ascribed to the damage caused by the insertion of a second electrode into the egg, which, since they were utilized to inject current, had very low resistance and large tips, causing a considerable loss of cytoplasm after removal. The remaining six eggs behaved differently. Two of them, although fertilized, showed no change in the membrane potential after the current injection ceased, remaining at the same RP value as that recorded before injection (see experiments 1 and 2 in Table 4). Two other eggs did not activate until the current injection had been discontinued and the potential had resumed its original resting value. Experiments 7 and 12 in Table 4 showed no response at all. All experiments using current injection are summarized in Table 4.

Table 4.

Current-induced depolarization in D. Pictus eggs

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

Schematic drawing of experiments where the membrane potential was depolarized by current injection. V = electrodes connected to the differential amplifier monitoring the transmembrane potential. I = currentinjection electrode. The top trace shows the response of the egg potential to current injection (bottom trace). ▴ ▴ = start and end of current injection. Δ = sperm added, ↑ = fertilization potential. Scale, Top trace, vertical bar = 10mV, horizontal bar = 10 s. Bottom trace, vertical bar = 100 mV, horizontal bar = 10 s.

Fig. 4.

Schematic drawing of experiments where the membrane potential was depolarized by current injection. V = electrodes connected to the differential amplifier monitoring the transmembrane potential. I = currentinjection electrode. The top trace shows the response of the egg potential to current injection (bottom trace). ▴ ▴ = start and end of current injection. Δ = sperm added, ↑ = fertilization potential. Scale, Top trace, vertical bar = 10mV, horizontal bar = 10 s. Bottom trace, vertical bar = 100 mV, horizontal bar = 10 s.

Polyspermy in Discoglossus pictus (Anura) eggs has been studied and the following new findings have emerged, (a) In comparison with other anuran amphibians, Discoglossus pictus eggs show a high incidence of polyspermy; (b) polyspermie eggs with less than five sperm develop normally; (c) susceptibility to polyspermy is a characteristic of the egg and does not depend on sperm; (d) polyspermie eggs have a normal fertilization potential; (e) sperm penetration is not regulated by the depolarization of the plasma membrane.

Fertilization in anurans is typically monospermic. The rate of polyspermy is very low and is incompatible with development. Elinson (1975) found only 4 polyspermie eggs from a total of 1200 Rana pipiens eggs. Picheral & Charbonneau (1982) reported from 500 eggs of Rana temporaria, Ranapipiens and Rana esculenta a 0 · 4% polyspermy rate. On the contrary, in vivo observations of Discoglossus pictus eggs have shown that a high percentage of polyspermy (33 · 5 %) is compatible with normal development. Recently Elinson (1987) found a low rate of polyspermy compatible with the normal development in the egg of the anuran Eleutherodactylus coqui.

Polyspermy in the present report on Discoglossus pictus eggs is not due to immaturity, since immature eggs do not generate normal FPs (Talevi et al. 1985). The polyspermie eggs studied here generated normal FPs and developed into normal tadpoles. Since polyspermie egg batches were always polyspermie when inseminated with different sperm batches, this does not depend on characteristics of the sperm but on intrinsic features of particular egg clutches.

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

In a previous paper (Talevi et al. 1985), Discoglossus pictus eggs at fertilization have been shown to generate a change in resting potential typical of anuran Amphibia (Maeno, 1959; Ito, 1972; Cross & Elinson, 1980; Grey et al. 1982; Schlichter & Elinson, 1981; Charbonneau et al. 1983α,6; Iwao et al. 1981; Webb & Nuccitelli, 1985).

It has been often been suggested (Cross & Elinson, 1980; Grey et al. 1982; Charbonneau et al. 1983α; Jaffe eí al. 1983; Webb & Nuccitelli, 1985) that the fast depolarization at fertilization in anuran eggs represents a fast block to polyspermy, similar to that reported for Echinodermata (Jaffe, 1976). The existence of a fast block 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 been shown in the Echinoderm egg of Lytechinus variegatus that opening of ion channels at fertilization precedes fusion of the sperm and egg plasma membrane (Hinkley et al. 1986; Longo et al. 1986). In Discoglossus pictus, polyspermie eggs show the same shift in voltage as monospermic eggs. In addition, experimental manipulation of the membrane potential before and during insemination has shown that interaction and penetration of the sperm are not related to membrane potential. In some cases, however, current injection may modify the membrane, disturbing the interaction between sperm and egg. Probably for this reason, in two of the cases reported here, fertilization only occurred when the current injection had ceased.

The question arises, what does regulate sperm entry? One possibility is the conformation and limited area of the animal dimple. In fact, fertilization cone formation and normal penetration can only occur in a restricted area of the dimple bottom called DI, with a diameter of about 200 μm (Talevi & Campanella, 1988). The fertilization cone has a diameter ranging from 40 to 70 μm and, therefore, the DI could accommodate about 5 – 8 cones. In vivo observations have shown that, in polyspermie eggs, the number of fertilization cones is generally 2 – 3. Only in one case were 8 fertilization cones observed. A further limitation to the number of sperm that can interact with this area is probably the particular characteristics of the plasma membrane, the animal plug and the other jelly layers surrounding the egg. In a previous paper (Talevi & Campanella, 1988), it has been shown that the animal plug plays an important role in directing the sperm towards the DI area. Since the animal plug is made of fibrillar material (Ghiara, 1960) one could hypothesize that the organization of such material gives rise to a series of set routes, through which only a limited number of sperm can progress to the vitelline coat. In polyspermie batches of eggs, this structure may be altered, facilitating multiple sperm entry.

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

The functional regionalization of the Discoglossus pictus egg, together with the observation that sperm entry in this species is independent of membrane potential, provides new evidence for alternative mechanisms of sperm – egg interaction.

I am grateful to Profs G. Ghiara, C. Campanella and B. Dale for suggestions and comments and to Mrs G. Falcone and G. Argenzio for photographic work. Supported by PhD program fund ‘Biología Evolutiva e del Differenziamento’ and to a CNR project on the Biology of Fertilization to G. Ghiara.

Campanella
,
C.
(
1975
).
The site of spermatozoon entrance in the unfertilized egg of Discoglossus pictus (Anura): An electron microscopy 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 study of the changes in the endoplasmic reticulum at activation
.
Devi Biol, (in press)
.
Charbonneau
,
M.
,
Moreau
,
M.
,
Picheral
,
B.
&
Guerrier
,
P.
(
1983a
).
Fertilization of amphibian eggs: A comparison of electrical responses between anurans and urodeles
.
Devi Biol
.
98
,
304
318
.
Charbonneau
,
M.
,
Moreau
,
M.
,
Picheral
,
B.
,
Guerrier
,
P.
&
Vilain
,
J. P.
(
1983b
).
Voltage noise changes during monospermic and polyspermie fertilization of mature egg of the anuran Rana temporaria
.
Dev. Growth Differ
.
25
,
485
494
.
Cross
,
N. L.
(
1981
).
Initiation of the activation potential by an increase 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 polyspermy in frogs mediated by changes in the membrane potential
.
Devi Biol
.
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 cortical contraction associated with egg activation in the frog Rana pipiens
.
Devi Biol
.
47
,
257
268
.
Elinson
,
R. P.
(
1986
).
Fertilization in amphibians: the ancestry of the block to polyspermy
.
Int. Rev. Cyt
.
101
,
59
100
.
Elinson
,
R. P.
(
1987
).
Fertilization and aqueous development of the Puerto Rican terrestrial-breeding frog, Eleutherodactylus coqui
.
J. Morph
.
193
,
217
224
.
Ghiara
,
G.
(
1960
).
Ricerche intorno alla struttura microscópica, submicroscopica ed istochimica e alie funzioni degli involucri ovulari di Discoglossus pictus Otth e di altre speccie di anfibi
.
Archo Zootal
.
45
,
9
92
.
Grey
,
R. D.
,
Working
,
P. K.
&
Hedrick
,
J. L.
(
1976
).
Evidence that the fertilization envelope blocks sperm entry in eggs of Xenops laevis: Interaction of sperm with isolated envelopes
.
Devi Biol
.
54
,
52
60
.
Grey
,
R. D.
,
Bastiani
,
M. S.
,
Wenn
,
D. J.
&
Schertel
,
E. R.
(
1982
).
An electrical block is required to prevent polyspermy in eggs 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
).
Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342
.
Devi Biol
.
118
,
148
154
.
Hope
,
J.
,
Humphries
,
A. A.
, Jr
&
Bourne
,
G. M.
(
1963
).
Ultrastructural studies on developing oocytes of the salamander Triturus viridescens
.
J. Ultrastr. Res
.
9
,
302
324
.
Ito
,
S.
(
1972
).
Effects of media of different ionic composition on the activation potential of anuran egg cells
.
Dev. Growth Differ
.
14
,
217
227
.
Iwao
,
Y.
(
1982
).
Differential emergence of cortical granule breakdown and electrophysiological responses during meiotic maturation of Toad oocytes
.
Dev. Growth Differ
.
24
,
467
477
.
Iwao
,
Y.
(
1985
).
The membrane potential changes of amphibian eggs during species and cross-fertilization
.
Devi Biol
.
111
,
26
34
.
Iwao
,
Y.
(
1987
).
The spike component of the fertilization potential in the toad, Bufo japonicus: changes during meiotic maturation and absence during cross-fertilization
.
Devi Biol
.
123
,
559
565
.
Iwao
,
Y.
,
Ito
,
S.
&
Katagiri
,
C.
(
1981
).
Electrical properties of Toad oocytes during maturation and activation
.
Dev. Growth Differ
.
23
,
89
100
.
Iwao
,
Y.
,
Yamasaki
,
H.
&
Katagiri
,
C.
(
1985
).
Experiments pertaining to the suppression of accessory sperm in fertilized Newt eggs
.
Dev. Growth Differ
.
27
,
323
331
.
Jaffe
,
L. A.
(
1976
).
Fast block to polyspermy in sea urchin eggs is electrically mediated
.
Nature, Lond
.
261
,
68
71
.
Jaffe
,
L. A.
,
Cross
,
N. L.
&
Picheral
,
B.
(
1983
).
Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians
.
Devi Biol
.
96
,
317
323
.
Jaffe
,
L. A.
&
Gould
,
M.
(
1985
).
Polyspermy-preventing mechanisms
.
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 electrophysiological studies of sperm-egg interaction of the sea urchin, Lytechinus variegatus
.
Devi Biol
.
118
,
155
166
.
Maeno
,
T.
(
1959
).
Electrical characteristics and activation potential of Bufo eggs
.
J. gen. Physiol
.
43
,
139
157
.
Mann
,
T.
,
Lutwak-Mann
,
C.
&
Hay
,
M. F.
(
1963
).
A note on the so-called seminal vesicles of the frog Discoglossus pictus
.
Acta Embryol. Morph, exp
.
6
,
21
.
N’Diaye
,
A.
,
Sandoz
,
D.
,
Bolsievieux-Ulrich
,
E.
&
Ozon
,
R.
(
1974
).
Action des androgènes chez lAmphibien Anoure Discoglossus pictus. IV. Etude ultrastructurale des phenomenes de secretion dans les vésicules seminales pendant la period l’accouplement
.
J. Microbiol
.
21
,
309
.
Nuccitelli
,
R.
&
Grey
,
R. D.
(
1984
).
Controversy over the fast, partial, temporary block to polyspermy in sea urchin: a réévaluation
.
Devi Biol
.
103
,
1
17
.
Nuccitelli
,
R.
,
Kline
,
D.
,
Busa
,
W.
,
Talevi
,
R.
&
Campanella
,
C.
(
1988
).
The activation current and intracellular calcium increase in the egg of the frog Discoglossus pictus
.
Devi Biol, (in press)
.
Okumoto
,
H.
(
1983
).
Studies on meioses in male hybrids and triploids in the Rana nigromaculata group. II. Auto and Allotriploids in Rana nigromaculata and Rana brevipoda
.
Sci. Rep. Lab. Amphibian Biol., Hiroshima Univ
.
6
,
183
205
.
Picheral
,
B.
(
1977
).
La fécondation chez le triton pleurodele. II. La penetration des spermatozoïdes et la reaction locale de loeuf
.
J. Ultrastruct. Res
.
60
,
181
202
.
Picheral
,
B.
&
Charbonneau
,
M.
(
1982
).
Anuran fertilization: A morphological reinvestigation of some early events
.
J. Ultrastr. Res
.
81
,
306
321
.
Runnstrom
,
J.
(
1961
).
The mechanism of protection of the eggs against polyspermy. Experiments on the sea urchin Paracentrotus lividus
.
Ark. Zool
.
13
,
565
571
.
Schlichter
,
L.
&
Elinson
,
R. P.
(
1981
).
Electrical responses of immature and mature Rana pipiens oocytes to sperm and other activating stimuli
.
Devi Biol
.
83
,
33
41
.
Talevi
,
R.
,
Dale
,
B.
&
Campanella
,
C.
(
1985
).
Fertilization and activation potentials in Discoglossus pictus (Anura) eggs: A delayed response to activation by pricking
.
Devi Biol
.
111
,
316
323
.
Talevi
,
R.
&
Campanella
,
C.
(
1988
).
Fertilization in Discoglossus pictus (Anura) I: Sperm-egg interactions in distinct regions of the dimple and occurrence of a late stage of sperm penetration
.
Devi Biol, (in press)
.
Webb
,
D. J.
&
Nuccitelli
,
R.
(
1985
).
Fertilization potential and electrical properties of the Xenopus laevis egg
.
Devi Biol
.
107
,
395
406
.
Whitaker
,
M. S.
&
Steinhardt
,
R. A.
(
1982
).
Ionic regulation of egg activation
.
Q. Rev. Biophys
.
15
,
593
666
.