Fertilization in Amphibians: The Ancestry of the Block to Polyspermy

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 101

Fertilization in Amphibians: The Ancestry of the Block to Polyspermy RICHARDP. ELINSON Department of Zoology, Universiry of Toronto, Toronto, Ontario, Canada Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fertilization of Anuran Eggs. . . . .................... A. Sperm Ent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Activation of the Egg . . . . . . , , . . . . . , . . . . . , . . . . . . . . . . . . . . C. The Block to Polyspenny . . . . . . . , . . . . . . . . D. Oocyte Maturation and the Block to Polysper E. Development of Polyspermic Anuran Eggs . . . . . . . . . . . . . . . . . 111. Fertilization of Urodele Eggs . . . , . , . . . , . , . . . . . . , . . . . . . . . A. Sperm Ent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Activation of the Egg . . . . . ................ ......... C. Development of Sperm and Egg Nuclei.. . . . . . . . . . . . . . . . . . . IV. Control of Accessory Sperm Nuclei in Urodele Fertilization . . . . . . . A. The Hypotheses of Bataillon and Fankhauser B. Androgenetic and Gynogenetic Development i C. A New Look at the Old Hypotheses.. . . . . . . . . . . . . . . . . . . . . . V. The Ancestral E g g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Comparison between Anuran and Urodele Fertiliz B. Blocks to Polyspermy in Fish .................... C. Hypothetical Ancestries. . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. I.

11.

59 60 61 65 69 71 13 16 16 78 79 80 80 82 85 87 87 89 90 93 94

I. Introduction During fertilization in mammals, echinoderms, and many other animals, a single sperm enters the egg, and all subsequent sperm are excluded. This pattern of fertilization is known as monospermy and depends on several blocks to polyspermy which the egg mounts after sperm entry. The mechanisms of the blocks to polyspermy have been carefully investigated in the last decade, and we have a reasonable picture of what is involved at the cellular and molecular levels. When the blocks are breached and more than one sperm enters, the polyspermic condition leads to death of the embryo. In contrast, birds, reptiles, and other animals exhibit physiological polyspermy. More than one sperm enters the egg, but only one sperm nucleus fuses with the egg nucleus, while the rest degenerate. The 59 Copyright d 1986 by Academic Preir. Inc. All rights of rcproduclion in any Tomi reserved.

60

RICHARD P. ELINSON

mechanism of this nuclear selection is not known and has barely been investigated in the last 40 years. Amphibians have both styles of fertilization. Monospermy is confined to the tailless frogs and toads which comprise the order Anura, while physiological polyspermy is found in the tailed newts and salamanders of the order Urodela. Nothing is known about fertilization in the wormlike apodans, the third living order of amphibians. At first glance, the occurrence of dissimilar fertilization mechanisms in two closely related groups of animals should provide an opportunity for the comparative study of the mechanisms. The opposite stance can also be taken; that is, the dissimilarity in fertilization mechanisms may indicate that the anurans and urodeles are only distantly related to each other. The question of relatedness of the anurans and the urodeles has been approached on embryological and anatomical grounds (Hanken, 1986). From embryological studies, the formation of the primordial germ cells (Nieuwkoop and Sutasurya, 1976, 1979), mesoderm (Smith and Malacinski, 1983), and notochord (Brun and Garson, 1984) appears to be different in the two orders. The primordial germ cells eventually give rise to the gametes, and because of their importance, Nieuwkoop and Sutasurya (1976, 1979) have argued that the anurans and urodeles are not closely related. They consider that the anurans and urodeles arose from different fishes and that modern amphibians have a diphyletic origin. Anatomical studies indicate important differences between the orders, but Parsons and Williams (1963) were able to find several anatomical characters which they believe linked the anurans and urodeles together in a monophyletic group. This conclusion has been discussed extensively (Jarvik, 1968; Estes and Reig, 1973; Stahl, 1974; Carroll and Currie, 1975; Gardiner, 1983), but unfortunately, there are no fossils which show the relationship between the two orders. The fossil record indicates that anurans and urodeles probably diverged from each other by the Triassic, 200 million years ago (Estes and Reig, 1973; Carroll, 1977), which predates the emergence of the modem orders of mammals. Therefore, even if the anurans and urodeles are derived from the same amphibian ancestor, they diverged from each other long ago. My objectives in this review are 2-fold. First, I will review the process of fertilization in amphibians, particularly as it relates to polyspermy. Second, I will assume that there was a common ancestral egg which gave rise to the anuran and urodele eggs and will discuss ways to explore the nature of this cytological ancestry. 11. Fertilization of Anuran Eggs

Anurans are commonly used for embryological studies, and fertilization in frogs and toads has been carefully investigated. Monospermic fertilization is

FERTILIZATION IN AMPHIBIANS TABLE I CLASSIFICATION OF ANUKANS AND

61

URODEI.F.S"

Order Anura Suborder Archaeobatrachia Superfamily Discoglossoidea Discoglossus p i c t ~ s Bombina , orientalis. Ascaphus truri. Alyres obstctricans Superfamily Pipoidea Xenopus laevis

Superfamily Pelobatoidea Suborder Neobatrachia Superfamily Bufonoidea Bufo umericunus, Bujo arenarum, Bufb bufo japonicus

Superfamily Microhyloidea Superfamily Ranoidea Rana pipiens. Rana temporaria (= Rana fusca)

Order Urodela Suborder Cryptobranchoidea Hynobius retardatus. Cryptobrunchus a1leganiensi.s

Suborder Sirenoidea Suborder Salarnandroidea Cynops pyrrhogaster, Notophthalmus viridescens (= Triturus viridesrens), Pleurodeles waltl. Triturus ulpestris. Trirurus palmatus, Triturus cristatus

Suborder Ambystomatoidea Ambysroma mexicanurn

"Anurans are classified according to Duellman (1975) and Laurent (1979) and the urodeles are classified according to Porter (1972). All species mentioned in the text are listed. Names in parentheses are the names used in the papers cited but which differ from the current species name.

characteristic of Xenopus laevis and many species of Rana and Bufo, and Wintrebert (1933) mentioned that only one sperm entered the egg of Discoglossus pictus. There are no reports of physiological polyspermy in anurans. The above animals represent four of the six superfamilies in the order Anura (Table I), so it is reasonable to conclude that monospermy is characteristic of anurans. In this section, the mechanisms of sperm entry and the block to polyspermy in anurans will be described. A. SPERMENTRY Fertilization of the anuran egg usually takes place externally in fresh water, although exceptions to this occur among terrestrial breeders (Townsend et al.,

62

RICHARD P. ELINSON

1981). At the time of fertilization, the egg is surrounded by the vitelline envelope which forms in the ovary and by several layers of jelly which are placed around the egg as it travels down the oviduct (Fig. 1). Numerous studies have shown that eggs without jelly are not fertilizable, but jellyless eggs can be fertilized in the presence of various jelly and oviducal extracts. This has proved easiest to do with various Bufo species (Katagiri, 1966, 1967, 1974; Barbieri and Raisman, 1969; Raisman and Pisano, 1970; Elinson, 1971a; Cabada et a f . , 1978), possible with Rana pipiens (Elinson, 1971a, 1973), but still very difficult with X . laevis (Stewart-Savage and Grey, 1984). The vitelline envelope is an important barrier to sperm since its digestion or removal greatly facilitates fertilization of jellyless eggs (Elinson, 1973; Katagiri, 1974; Stewart-Savage and Grey, 1984). The experiments with jellyless eggs have helped to define the role of the oviducal secretions in fertilization, and two activities appear to be involved, the first affecting the vitelline envelope and the second involving the jelly itself. The vitelline envelope is altered as the egg passes through the initial non-jellysecreting part of the oviduct called the pars recta (Fig. l ) , and this alteration is important for later sperm penetration (Grey et al., 1977; Cabada et al., 1978; Miceli et al., 1978a; Yoshizaki and Katagiri, 1981). Miceli and co-workers have

FIG. I . The frog reproductive system. The egg is released from the ovary into the coelom. The coelomic egg, surrounded by a vitelline envelope (ve) but lacking jelly, is moved by cilia to the opening of the oviduct. After passing through the non-jelly-secreting pars recta, the oviducal egg begins to acquire jelly. During amplexus with a male, the egg with several jelly coats is released to the outside where it is inseminated (sperm are not to scale). (The ovaries have been pulled to the sides in this frog, redrawn from Walker, 1967.)

FERTILIZATION IN AMPHIBIANS

63

characterized a proteinase from the pars recta which acts on the vitelline envelope, and they consider that the limited digestion of the envelope makes it penetrable by sperm (Miceli et al., 1978b, 1980; Miceli and Fernindez, 1982). On the other hand, Katagiri et a f . (1982) have presented evidence that a pars recta secretion induces the acrosome reaction of the sperm, and in this way, the secretion is important for sperm penetration. The relative roles of these two possible activities are yet to be determined. Treatment of eggs with pars recta secretions is necessary but not sufficient to allow fertilization. The treated eggs must also be inseminated in the presence of extracts from the jelly itself (Cabada et al., 1978; Miceli et al., 1978a; Katagiri et al., 1982). What these jelly extracts are doing is not known, although Ishihara et al. (1 984) have recently made the intriguing observation that a medium mimicking the salt content of the jelly extract can support fertilization. This result suggests that the jelly may function by providing an ionic microenvironment required for the acrosome reaction and other events of fertilization. Besides helping sperm to fertilize the egg, the jelly provides an absolute as well as a relative limit on sperm access to the egg. With respect to the absolute limit, the jelly hydrates rapidly upon exposure to low-ionic-strength solutions such as pond water, and forms a barrier which prevents fertilization at times ranging from 15 minutes to 1 hour after laying (Katagiri, 1961; Barbieri and Villeco, 1966; Elinson, 1971b; Wolf and Hedrick, 1971; del Pino, 1973). With respect to the relative limit, the number of sperm which normally reach the egg surface may be small. The best test of this limit would be to see how many sperm enter eggs during natural matings and under conditions where the block to polyspermy is inhibited. While this test has not been done, artificial insemination of immature eggs which lack a block to polyspermy (Section 11,D)gave average numbers of entries of 3 for R . pipiens (Schlichter and Elinson, 1981) and 19 for B . bufo japonicus (Katagiri, 1974). Artificial insemination of mature eggs, with a partial inhibition of their block to polyspermy by iodide (Section ll,C), gave 26 entries per egg in R . temporaria (Charbonneau et al., 1983a) and only 40% polyspermy in R . pipiens (Cross and Elinson, 1980). Natural mating of X . laevis in the presence of iodide yielded 70% polyspermy with an average of 5.6 sperm per egg (Grey et al., 1982). This information suggests that the number of sperm which reach the egg surface and can enter the egg is relatively small. As will be seen later, this low number of sperm is not much different from the number of sperm entries which occur during physiological polyspermy in urodeles (Section 111,A).

The sperm normally take 3-8 minutes to swim through the jelly (Elinson, 1975; Stewart-Savage and Grey, 1982) during which time they undergo the acrosome reaction characteristic of most animal sperm (Raisman and Cabada, 1977; Raisman et al., 1980; Yoshizaki and Katagiri, 1982). The acrosome reaction releases a protease, usually a trypsinlike enzyme, which enables the sperm

64

RICHARD P. ELINSON

to mount an enzymatic attack against the vitelline envelope (Elinson, 197 I b, 1974a; Penn and Gledhill, 1972; Raisman and Cabada, 1977; Iwao and Katagiri, 1982). Sperm approach any part of the egg surface in R . pipiens (Elinson, 1975), and except for D. pictus, with its unusual jelly arrangement (Wintrebert, 1933; Campanella, 1975), there is no indication in other species of restrictions on sperm approach to the egg. Nonetheless, the single, fertilizing sperm always enters the pigmented animal half of the egg and never the yolky vegetal half (Newport, 1853; Hertwig, 1877; Born, 1885; Roux, 1887; Subtelny and Bradt, 1963; Katagiri, 1974; Elinson, 1975; Schlichter and Elinson, 1981; Grey et a f . , 1982; Charbonneau and Picheral, 1983; Charbonneau et a f . , 1983a). The basis for the restriction of sperm entry to the animal half is unknown, although the animal and vegetal surfaces differ in significant ways. Both surfaces are covered with microvilli, but the microvilli in the animal half are much longer than those in the vegetal half (Elinson, 1980; Charbonneau and Picheral, 1983; Dictus et a f . , 1984). The plasma membrane of the animal half has slightly more small intramembranous particles (Bluemink and Tertoolen, 1978) and significantly less lateral mobility of lipids (Dictus et a f . , 1984) compared to that of the vegetal half. It would be interesting to see whether alteration of these surface or membrane properties affects sperm entry. The entry of the sperm into the egg and the subsequent changes at the site of entry have been followed by scanning electron microscopy (Elinson and Manes, 1978; Picheral and Charbonneau, 1982; Charbonneau and Picheral, 1983; G6mez and Manes, 1984). The sperm enters with its anterior tip first, an orientation expected from the anterior location of the acrosome. A small body called the fertilization body forms at the site of entry which later is either sloughed off or transformed into a distinct clump of microvilli which persists for several hours. Besides this surface scar, the area of entry can later be recognized by a pigment accumulation variously called the sperm entry site, the sperm entry point, or the paternal streak. This pigment accumulation is visible on lightly pigmented eggs such as those of X . faevis (PaleCek et a f . , 1978; Stewart-Savage and Grey, 1982) and can be revealed on darkly pigmented eggs by bleaching the cortical pigment (Elinson, 1975). The pigment accumulations are probably due to the activities of the sperm-aster, and they serve as useful markers for experiments on polyspermy and embryonic axis formation. With respect to species specificity of fertilization, most crosses within a genus are possible, as well as some crosses between genera or even between orders (Montalenti, 1938; Moore, 1955; Blair, 1972; Subtelny, 1974). In two cases, interspecific fertilization failures were overcome by passing the egg of the first species down the oviduct of the second species, where the egg acquired the jelly coat. In the cross X . borealis female X X . faevis male, X . faevis sperm fail to penetrate the X . borealis innermost jelly layer. The cross is successful when the X . borealis egg is first covered with X . faevis jelly (Brun and Kobel, 1977). In

65

FERTILIZATION IN AMPHIBIANS

crosses involving R. clamitans, the sperm of R. clamitans can fertilize its own eggs as well as eggs of other Rana, but the sperm of other Rana cannot fertilize eggs of R . clamitans. The sperm of R. clamitans have a very high level of acrosomal, proteolytic activity, which probably accounts for its fertilizing ability (Elinson, 1974a). Sperm of R. pipiens can fertilize R. clamitans eggs when the eggs are enrobed in R. pipiens jelly. The oviducal secretion from R. pipiens may alter the vitelline envelope of the R . clamitans egg (Elinson, 1974b), similar to the postulated activity of the pars recta.

B. ACTIVATION OF THE EGG The unfertilized anuran egg is arrested at Metaphase I1 of meiosis and can be activated either by a sperm or by a number of artificial stimuli such as a needle prick, an electric shock, or the ionophore A23187. The initial responses of the egg to an activating stimulus can be detected electrophysiologically, and these events are followed by a series of morphological changes (Fig. 2). Within 1 second of stimulation, there is a depolarization of the egg membrane, and this change in membrane potential is called the activation or fertilization potential Time (minutes)

1

3-

4-

(,

Cortical granule exocytosis --+Fertilization

Cortical contraction

envelope

Microtubule collapse

5.

5-

6-

FIG.2. Time course of activation events. cg, Cortical granules; ve, vitelline envelope; mv, microvilli; F, F layer. In the diagram of cortical granule exocytosis, the sperm and the curvature of the egg are exaggerated relative to the vitelline envelope and the cortical granules.

66

RICHARD P. ELINSON

(Maeno, 1959; Ito, 1972; Cross and Elinson, 1980; Cross, 1981; Schlichter and Elinson, 1981; Iwao e t a f . , 1981; Iwao, 1982; Grey et al., 1982; Charbonneau et al., 1983a,b; Jaffe et a f . , 1983; Webb and Nuccitelli, 1985). The depolarization is due primarily to the opening of chloride channels, measurable as a decrease of the membrane resistance, and chloride ions leave the egg (Maeno, 1959; Ito, 1972; Cross, 1981; Charbonneau e t a f . , 1983b; Jaffe and Schlichter, 1985; Webb and Nuccitelli, 1985). Potassium ions also leave the egg at activation (Jaffe and Schlichter, 1985), and both the potassium and chloride channels open in a wavelike pattern which propagates over the egg surface (Jaffe et a f . , 1985; Kline and Nuccitelli, 1985). About 10 seconds after the start of the activation potential, the membrane capacitance increases, indicating an increase in membrane surface area and signaling the onset of cortical granule exocytosis (Jaffe and Schlichter, 1985; Peres and Bernardini, 1985). The cortical granules, first observed by Motomura (1952), lie just under the plasma membrane before activation and fuse to it upon activation, releasing their contents to the outside. Cortical granule exocytosis occurs as a wave from the point of sperm entry or from a site of localized activation, such as when the egg is pricked with a needle (Balinsky, 1966; van Gansen, 1966; Kemp and Istock, 1967; Kas’yanov et al., 1971; Grey et a f . , 1974; Campanella and Andreuccetti, 1977; Hara and Tydeman, 1979; Goldenberg and Elinson, 1980; Picheral and Charbonneau, 1982; Charbonneau and Picheral, 1983; Takeichi and Kubota, 1984; G6mez et al., 1984). The wave of exocytosis passes more rapidly across the pigmented animal half of the egg than across the yolkier vegetal half and takes about 2 minutes for completion. Shortly after cortical granule exocytosis, the egg undergoes a series of structural changes. The exocytosis is accompanied by a reorganization of the egg cortex, as indicated by an elongation of the microvilli after the exocytotic wave passes (Balinsky, 1966; Goldenberg and Elinson, 1980; Picheral and Charbonneau, 1982) and the appearance of dense cortical cytoplasm, which excludes organelles (Grey et al., 1974; Campanella and Andreuccetti, 1977). The cortex becomes coherent and can be dissected from the egg as an elastic sheet (Elinson, 1983). These changes may reflect the polymerization of actin as seen with activation of sea urchin eggs (Vacquier, 1981), but biochemical studies on the amphibian egg cortex (Richter, 1980, 1983) have not been directed at this question. A series of surface contraction waves sweeps over the egg (Takeichi and Kubota, 1984; Kline and Nuccitelli, 1985) and the cortex contracts toward the animal pole, reducing the surface area covered by the pigmented cortex (Elinson, 1975; Stewart-Savage and Grey, 1982). The cortical contraction is maximal at about 5 minutes, and then the cortex slowly relaxes. At about the time that the cortex is contracting, the cytoplasm changes from a firm consistency to a fluid one (Elinson, 1983). This change in consistency probably reflects a change in the egg cytoskeleton, and it is correlated with a massive depolymerization of micro-

67

FERTILIZATION IN AMPHIBIANS TABLE I1 TIMING OF EVENTSIN THE FIRSTCELL

CYCLE"

Species A.

Events Formation of second polar body Close association of male and female pronuclei Metaphase First cleavage

X . laevis

R . pipiens

mexicanum

P . waltl

N. viridescens

0.16-0.25

0.17

0.14

0.19

0.1

0.45

0.6

0.5

0.50

0.5

0.85 1 .o

0.8

0.9 I .o

0.91 1.O

0.8

(6 hours at

(-8 hours at 20")

(1.5 hours at 18")

I .o (3 hours at 18")

(7 hours at 18")

18")

I .o

OThe time scale is normalized by setting insemination at 0 and first cleavage at 1.0. Data are from Ubbels et al., 1983 (X.luevis); Subtelny and Bradt, 1963 (R. pipiens); Wakimoto, 1979 ( A . mexiranum); Labrousse, 1959 (8'. wuirl); and Fankhauser and Moore, 1941a (N.viridesrertsl.

tubules (Elinson, 1985). All of these morphological changes, along with the activation potential and cortical granule exocytosis, constitute a group of early events of activation. Several later morphological features are commonly used as signs of activation. These include egg rotation, second polar body release, grey crescent formation, and cleavage. The exocytosis of the cortical granules leads to the formation of the fluid-filled perivitelline space between the egg and the forming fertilization envelope (Section II,C), and this permits the rotation of the egg with respect to gravity. The rotation places the pigmented animal half up and the nonpigmented vegetal half down, which is the same defensive color pattern that fish present to would-be predators. Failure of egg rotation leads to abnormal development due to gravity-driven rearrangements of the cytoplasm (Neff et al., 1983). Second polar body formation, indicating the completion of meiosis, occurs about one fifth of the way through the first cell cycle (Table II), and grey crescent formation, marking the acquisition of embryonic dorsoventral polarity, starts about halfway through the cycle (Manes and Elinson, 1980). Fertilized eggs of most anurans studied cleave at 1.5-3.5 hours after fertilization at 18°C. Artificially activated eggs show abortive cleavages somewhat later than the time of first cleavage in fertilized eggs (Sambuichi, 1981). A number of studies have indicated that an increase in free Ca2 is involved in activation of the frog egg, and Ca2 may be the primary trigger for activation. Eggs can be activated by the calcium ionophore A23 187 (Steinhardt et al., 1974; Belanger and Schuetz, 19751, and activation by pricking with a needle depends +

+

68

RICHARD P. ELINSON

on the level of external calcium (Wolf, 1974a; Goldenberg and Elinson, 1980). Both of these activators cause a leak of calcium into the egg. Eggs of X . faevis can also be activated by the injection of inositol trisphosphate, which, as in other cells, causes the release of Ca2+ (Busa and Nuccitelli, 1985; Busa et al., 1985; Kline and Nuccitelli, 1985; Picard er al., 1985). Measurements with either aequorin (Wasserman et al., 1980) or Ca2 -selective microelectrodes (Busa and Nuccitelli, 1985) show an increase in free Ca2+ at the time of activation. In addition, Ca2+ is involved in each of the events constituting activation. The fertilization potential is due to an efflux of chloride ions, which is due in turn to the opening of chloride channels by Ca2+ (Cross, 1981; Young et a f . , 1984). Cortical granules break down in vivu or in virru when exposed to Ca2+ (Hollinger and Schuetz, 1976; Hollinger et af., 1979; Goldenberg and Elinson, 1980), and the cortical contraction occurs in response to A23187 or Ca2+ in either unactivated or activated eggs (Schroeder and Strickland, 1974; Merriam and Sauterer, 1983; Christensen er al., 1984). Microtubules are sensitive to Ca2+, which may account for their disappearance at the time of activation (Elinson, 1985). Finally, cytostatic factor, which is responsible for the arrest at Metaphase 11, is inactivated by Ca2 , thus allowing meiosis to resume (Meyerhof and Masui, 1977). Besides the increase in free Ca2+, there is a transient acidification of the cytoplasm following activation (Webb and Nuccitelli, 198I ) , but the functional significance of this change in pH is not known. The increased level of free Ca2 probably travels as a wave around the egg as indicated by the wave of cortical granule exocytosis. The propagation of the Ca2+ wave in X. laevis appears to involve a special membrane system in the cortex called the cortical endoplasmic reticulum. The cortical endoplasmic reticulum surrounds the cortical granules (Grey et al., 1974) and forms an extensive membrane network in the cortex (Campanella and Andreuccetti, 1977). The cortical endoplasmic reticulum forms junctions with the plasma membrane, causing Gardiner and Grey (1983) to remark on its similarity to the sarcoplasmic reticulum, which regulates Ca2 in muscle. Two lines of evidence suggest that the cortical endoplasmic reticulum is involved in propagation of the Ca2 signal around the egg. First, the egg acquires the ability to undergo propagated cortical granule exocytosis during oocyte maturation. This ability is acquired at the same time that the cortical endoplasmic reticulum forms (Charbonneau and Grey, 1984; Campanella et al., 1984). Second, the cortical endoplasmic reticulum changes following activation of the egg, consistent with its possible role in the activation process (Campanella and Andreuccetti, 1977; Gardiner and Grey, 1983). Ca2+ is found to be associated with the membranes of the cortical endoplasmic reticulum, but only after activation has started (Andreuccetti et al., 1984). The changes in free and bound Ca2+ relative to the cortical endoplasmic reticulum need to be better defined in order to understand how Ca2 movement is regulated in the cortex. The various events of fertilization and activation all show important dif+

+

+

+

+

+

FERTILIZATION IN AMPHIBIANS

69

ferences related to the animaUvegeta1 polarity of the egg. I have already discussed the restriction of sperm entry to the animal half and the differences in plasma membrane between the animal and vegetal halves. Cross (1981) found that activation potentials were significantly slower following iontophoresis of Ca2+ into the vegetal half as compared to the animal half. Goldenberg and Elinson ( 1980) noted that the wave of cortical granule exocytosis traveled more slowly through the vegetal half than the animal half and more Ca2+ was required to prick-activate an egg in the vegetal half. Finally, Gardiner and Grey (1983) saw fewer junctions between the cortical endoplasmic reticulum and the plasma membrane of the vegetal half compared to the animal half. The sum of these results indicates that activation events are more difficult to initiate and they propagate more slowly in the vegetal half, probably because of a difference involving Ca2 regulation. +

C. THE BLOCKTO POLYSPERMY The anuran egg has a dual system to ensure that fertilization is monospermic. The fertilization potential provides a fast, transient block to polyspermy, while the cortical granules contribute to a slow, permanent block. Upon fertilization, the membrane potential shifts from a negative potential to a positive one within 1 second and returns to a negative value 10-30 minutes later (Cross and Elinson, 1980; Schlichter and Elinson, 1981; Iwao et al., 1981; Grey et al., 1982; Charbonneau et al., 1983b; Jaffe et al., 1983). The event which triggers the fertilization potential is not known, but the potential change occurs within a few seconds of effective sperm-egg interaction. This timing has been shown by blocking sperm entry with an injection of current into the egg and seeing how soon a fertilization potential occurs after the blocking current is turned off (Cross and Elinson, 1980; Charbonneau et al., 1983b; Jaffe et al., 1983). The fertilization potential is one of the earliest detectable events associated with sperm entry. It results from opening of ion channels, measured as a change in membrane conductance, and precedes a change in the membrane capacitance (Jaffe and Schlichter, 1985). Charbonneau et al. (1983a) have reported an increase in voltage noise prior to the fertilization potential, but the cause and significance of this measurement is unknown. Experimental evidence that the fertilization potential serves as a block to polyspermy has been presented for R . pipiens, R . temporaria, B. americanus, and X . laevis. The evidence consists of complementary results. On the one hand, sperm are unable to enter the egg when the membrane potential is held positive by current injection (Cross and Elinson, 1980; Charbonneau et al., 1983b; Jaffe et al., 1983). On the other hand, polyspermy results when the normal depolarization is prevented by a high external concentration of iodide or other halides (Bataillon, 1919; Cross and Elinson, 1980; Grey et a/.,1982; Charbonneau et al., 1983a.b). These ions prevent the membrane depolarization by moving into

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RICHARD P. ELINSON

the egg when the chloride channels open (Young et al., 1984). The external ion concentration must be high enough to affect the depolarization but low enough to permit sperm motility. The fertilization potential indicates that the egg membrane has changed, but how this change affects sperm entry is unknown for frogs or for any other animal. One hypothesis proposed by Jaffe et al. (1983) is that the sperm uses an insertion protein to fuse with the egg. The charge on the insertion protein compared to that at the egg membrane may determine whether insertion and subsequent gamete fusion is possible. The membrane potential returns to negative values within 30 minutes of sperm entry, so a more permanent block to polyspermy is required. This block is provided by the contents of the cortical granules. Upon release outside the egg, the contents interact with the vitelline envelope and the jelly to produce the fertilization envelope, which blocks sperm entry (Fig. 2). This story was reviewed by Schmell et al. (1983), and I will describe the main features here. Upon transformation of the vitelline envelope to the fertilization envelope, it becomes resistant to solubilization by temperature, chemicals, and proteolytic enzymes (Katagiri, 1963; Wolf, 1974b; Wolf et al., 1976; Miceli et al., 1977). The fertilization envelope is not digested by a sperm lysin (perhaps the acrosomal protease) in B . arenarum (Raisman and Barbieri, 1969; Miceli et al., 1977), and sperm are unable to penetrate isolated fertilization envelopes in X . laevis (Grey et al., 1976). The block to polyspermy produced by the fertilization envelope is in place about 4-6 minutes after the first sperm reaches the egg (Grey et al., 1982). The simplest hypothesis based on these results is that the sperm cannot reach the egg once the fertilization envelope forms, since its acrosomal protease is ineffective in digesting a hole in the envelope. The transformation of the vitelline envelope to the fertilization envelope has been examined in X . laevis primarily by Hedrick and co-workers. They found that a cortical granule lectin passes through the vitelline envelope and interacts with galactose residues in the innermost jelly layer (Wyrick et al., 1974). The cortical granule lectin has been localized in the cortical granules (Greve and Hedrick, 1978), but the possibility has been raised that its ligand is found in a prefertilization layer secreted by pars recta and not in the inner jelly layer (Yoshizaki and Katagiri, 1984; Yoshizaki, 1984). In either case, the lectinligand interaction produces the electron-dense fertilization layer (F layer) at the vitelline envelope-jelly interface (Grey et al., 1974; Wolf, 1974b; Yoshizaki and Katagiri, 1984). The F layer traps the contents of the cortical granules between the fertilization envelope and the egg plasma membrane. As a result of this osmotic barrier, water flows into the perivitelline space, and the fertilization envelope is raised from the egg plasma membrane (Nishihara and Hedrick, 1977; Schmell et al., 1983). In addition to the formation of the F layer, a few glycoproteins in the vitelline envelope undergo a limited hydrolysis (Wolf et al., 1976; Schmell et al., 1983).

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This change is correlated with the reduction in solubility and trypsin sensitivity of the fertilization envelope, with the F layer playing little or no role (Wolf, 1974b; Schmell et al., 1983). This result raises the question as to whether the F layer or another alteration of the vitelline envelope, such as hydrolysis, is important in the block to polyspermy. Although the presence of the F layer is correlated with blocking sperm penetration (Wyrick et al., 1974), work with isolated fertilization envelopes provides evidence for a role of alteration of the vitelline envelope itself (Grey et al., 1976). At this time, it may be worthwhile to consider a dual hypothesis. First, the alteration of the vitelline envelope may reduce its sensitivity to sperm acrosomal protease, decreasing the probability of sperm penetration. Second, the F layer may represent an inactivation by precipitation of a pars recta component important in gamete interaction. It would be worth seeing whether a function such as sperm binding or induction of the acrosome reaction can be attributed to the prefertilization layer from the pars recta. The analysis of the role of cortical granule components in the block to polyspermy would be aided by a method for specifically inhibiting cortical granule exocytosis, but this method has not yet been found. Exocytosis can be prevented by poisoning eggs with CO, (Bataillon and Tchou Su, 1930; Goldenberg and Elinson, 1980), but CO, appears to block all activation events except for the increase in membrane conductance associated with the fertilization potential (Peres and Bernardini, 1985). D. OOCYTEMATURATION AND

THE

BLOCKTO POLYSPERMY

The fully grown amphibian oocyte is found in the ovary with its large nucleus, the germinal vesicle, in Prophase I of meiosis. Upon stimulation with hormone, the immature oocyte begins maturation, and the oocyte is ovulated into the body cavity or coelom (see Masui and Clarke, 1979). From the coelom, the maturing oocytes are moved into the oviduct, where they acquire the jelly necessary for fertilization (Fig. 1). During maturation, the nucleus continues through meiosis until it arrests at Metaphase 11. At about the time that Metaphase I1 is achieved, the oocyte is considered mature in both a nuclear and a cytoplasmic sense. The oocyte becomes activatable and can generate a propagated response, including the block to polyspermy, when pricked with a needle or when fertilized. Prior to maturity, insemination leads to polyspermy . The youngest immature oocytes with jelly are usually at about Metaphase I, and oocytes at this stage have been used most often for experiments (Bataillon, 1929; Bataillon and Tchou Su, 1930; Tchou Su and Chen Chou Hsi, 1942; Tchou Su and Wang Yu-Lan, 1964; Katagiri, 1974; Elinson, 1977; Schlichter and Elinson, 1981). The many sperm which enter these oocytes usually form small sperm spindles, reflecting the cytoplasmic conditions of the oocyte (Bataillon, 1929; Bataillon and Tchou Su, 1930, 1934; Elinson, 1977). It should be possible to inseminate oocytes at earlier

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meiotic stages by initiating maturation in vitro of oocytes freed from the ovarian follicles and inseminating them in the presence of jelly extracts, but this has not yet been done. The maturing oocytes can be used to examine the development of the block to polyspermy mechanisms, and both the slow block, involving cortical granules, and the fast block, involving ion channels, have been examined in this way. The fully grown ovarian oocyte has cortical granules which formed from the endoplasmic reticulum while the oocyte was growing (Wishnitzer, 1966; Dumont, 1972). Activating stimuli such as 1 pA4 ionophore A23187, electric shock, or a needle prick do not initiate cortical granule exocytosis. The cortical granules do respond, however, to 10 pA4 A23187 (Iwao, 1982) or to a direct injection of Ca2+ (Hollinger and Schuetz, 1976; Hollinger et al., 1979), implying that the cortical granules themselves are Ca2 sensitive. The ability of cortical granules to respond to 1-2 pA4 A23187 develops midway through maturation and probably after germinal vesicle breakdown. The ability to respond to A23187 precedes by several hours the ability of the egg to undergo a complete propagated response to pricking (Belanger and Schuetz, 1975; Iwao, 1982; Charbonneau and Grey, 1984; Campanella et al., 1984). The Ca2+ sensitivity of cortical granules tested in vitro does not differ between cortical granules isolated from Metaphase I oocytes which do not respond to pricking and those isolated from responsive Metaphase I1 oocytes (Goldenberg and Elinson, 1980). These results indicate that the later acquisition of the response to pricking is not due to a change in the cortical granules themselves. As discussed in Section II,B, the ability to propagate a wave of cortical granule exocytosis is correlated with the formation of a cortical endoplasmic reticulum (Charbonneau and Grey, 1984; Campanella et al., 1984). If the cortical endoplasmic reticulum is able to sequester and release Ca2+ as hypothesized, its formation would be a sufficient explanation for the onset of the oocytes’ ability to propagate the wave of cortical granule exocytosis. During maturation, the oocyte also acquires the ability to produce the fertilization potential. Treatment of B . bufo Metaphase I oocytes with A23187 produced a relatively small, slow depolarization, with little change in membrane resistance. As the oocytes matured, the change in potential became larger and faster (Iwao, 1982). Similarly, pricking of oocytes of R. pipiens or B . bufo japonicus at stages between Metaphase I and Metaphase I1 gave a depolarization which is smaller in magnitude than that found in mature eggs (Schlichter and Elinson, 1981; Iwao et al., 1981). When Metaphase 1 oocytes were inseminated, the entry of each sperm caused a transient depolarization, and the potential returned to the original resting level within 30 seconds (Schlichter and Elinson, 1981). The difference between the response of the immature oocyte and the mature one is not in the ability of the membrane to depolarize but in the inability of the immature oocyte to sustain the depolarization. Although it is likely that the +

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depolarization is due to the opening of C1- channels as in the mature egg, the ionic basis has not been determined for either depolarization or repolarization in these oocytes. The examination of maturing oocytes permits the testing of interrelationships between the events of maturation and the block to polyspermy as well as between the different events of the block to polyspermy. For instance, the block to polyspermy mechanisms appears after germinal vesicle breakdown, suggesting a possible requirement for the germinal vesicle. The germinal vesicle is not needed, however, since following its removal, oocytes produce a fertilization potential (Iwao et al., 1981), undergo cortical granule exocytosis (Smith and Ecker, 1969; Skoblina, 1969), and are monospermic (Katagiri and Moriya, 1976). With respect to the events of the block to polyspermy, experiments on oocyte maturation show that the addition of cortical granule membrane to the egg plasma membrane via exocytosis is not the basis of the fertilization potential, since A23187 causes oocytes to undergo cortical granule exocytosis before it causes them to show a change in membrane potential (Iwao, 1982). This and electrophysiological evidence (Jaffe and Schlichter, 1985; Peres and Bernardini, 1985) suggest that the C1- channels are in the plasma membrane rather than in the cortical granule membrane. Finally, both cortical granule exocytosis and the fertilization potential require Ca2 , so the ability of the egg to propagate a wave of exocytosis and the type of membrane potential change may be correlated. These relationships require further clarification. After achieving maturity, the egg has the ability to mount an efficient block to polyspermy, but this ability is lost within hours or days, depending on the species. Overmature eggs are also polyspermic (Bataillon and Tchou Su, 1934; Wakahara et al., 1984), but the cause of this is not known. No one has investigated the overmature eggs in terms of the fertilization potential, the cortical granules, or the other events of activation. +

E. DEVELOPMENT OF POLYSPERMIC ANURAN EGGS Although fertilization of mature eggs is normally monospermic, it is possible to induce polyspermy experimentally and to ask how the egg develops. The analysis of polyspermic eggs was performed by Brachet (1910a,b; see 1912 for review) and Herlant (1911), and their work remains the best description. In general, polyspermic eggs cleave at about the same time as monospermic ones (Brachet, 1912) or slightly earlier (Charbonneau et ul., 1983a). They usually have multiple furrows rather than one, and the number of furrows is dependent on the number of entering sperm (Fig. 3). Herlant (191 1) analyzed dispermic and trispermic R. temporaria eggs, while Brachet (1910a,b) described R. temporuriu eggs with greater degrees of polyspermy. In Herlant’s cases, each sperm nucleus developed an aster, and the asters excluded each other. As a result, the sperm

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Frog

Newt

FIG.3 . Comparison between a trispermic egg in a frog and in a newt. In the frog, each sperm sets up an independent spindle, two of which contain only sperm chromosomes and one of which has sperm and egg chromosomes. Cleavage furrows bisect each spindle, producing three blastomeres each with two nuclei. In the newt, a spindle is set up only by the combined sperm and egg nucleus while the two other sperm nuclei form a monaster. A single cleavage furrow divides the egg into two blastomeres, each containing a diploid nucleus. The accessory nuclei degenerate.

nuclei never fused with each other but remained in the center of their aster, more or less equally spaced throughout the animal cytoplasm. The female nucleus entered into one of the asters and fused with the male pronucleus therein. At the first mitosis, spindles were formed for each sperm nucleus, and as the nucleus divided, a cleavage furrow formed (Fig. 3). Since each spindle generates a furrow, multiple furrows formed, characteristic of the number of sperm pronuclei and spindles present. In the dispermic egg, a single vertical furrow formed, indistinguishable from the furrow in a monospermic egg. This furrow actually represents the fusion of two half-furrows, each generated by a separate spindle, and each of the two blastomeres contains two nuclei, one haploid and one diploid. The second cleavages are again vertical and the egg is divided into six cells. Two cells contain a haploid nucleus, two cells contain a diploid nucleus, and two cells contain two nuclei, one haploid and one diploid. In the trispermic egg, three vertical furrows form, dividing the egg into three cells, and

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again each cell contains two nuclei, two with a haploid and a diploid nucleus and one with two haploid nuclei (Fig. 3). With levels of polyspermy up to 10 sperm, the number of furrows is equal to the number of sperm. After this point, there are fewer furrows produced, relative to the number of sperm (Brachet, 1910a,b, 1912). A likely explanation for this behavior is that each sperm controls a limited area of cytoplasm, as defined by its aster. With more sperm, each sperm controls less cytoplasm until the spindle it can produce is too small or too removed from the surface to induce a cleavage furrow. An alternative explanation is that there is a limitation of cytoplasmic components necessary for the transformation of the sperm nucleus and its progression through the cell cycle. Such a limitation has been suggested for the much smaller mouse egg (Czolowska et a / . , 1984). The patterns found by Herlant (19 1 1) for R . temporaria are similar to those for X . laevis, with some interesting additions (Render and Elinson, 1986). As in the monospermic case, pigment accumulates around the sperm entry sites. This movement of pigment produces distinct unpigmented white stripes between sperm entry sites, so in a dispermic egg there is one white stripe, while in a trispermic one there is a Y-shaped white area. When cleavage occurs, the furrow cuts the pigmented areas in half, so that in a dispermic egg, the first furrow, consisting of two half-furrows, is perpendicular to the white stripe. The second set of furrows of a dispermic egg tends toward the horizontal, producing a fourcell embryo. The difference in the second cleavage plane between X . laevis and R . temporaria probably reflects differences in the content of cytoplasm and yolk. The essential feature of the behavior of sperm in a polyspermic anuran egg is that each one develops fully and independently. Each sperm produces an aster, a bipolar spindle on which the chromosomes divide, and a cleavage furrow oriented according to the position of the spindle (Fig. 3). The nuclei in the resulting binucleate cells do not fuse, and each acts independently to produce the next set of furrows. This pattern of cleavage yields a genetic mosaic in which some cells are haploid, some are diploid, and some have an undetermined but abnormal number of chromosomes. This situation in frogs differs markedly from the pattern in sea urchins, whose analysis by Boveri (1902) was essential in demonstrating the qualitative importance of individual chromosomes. In the dispermic sea urchin egg, a tripolar or tetrapolar spindle forms, leading to an unequal distribution of chromosomes in the resulting three or four cells. Since very few of these embryos developed, Boveri concluded that the types of chromosomes rather than the quantity of chromatin were necessary for normal development. With the genetic mosaics produced in the frog example, dispermic and even some higher polyspermic eggs are able to develop. In R . temporaria, the survival to larvae of di- and trispermic eggs was on the order of 10% (Brachet, 1912), with one surviving for three months (Herlant, 191 1). In X . laevis, 70% of di- and

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trispermic embryos gastrulated and formed axial structures (Render and Elinson, 1986).

111. Fertilization of Urodele Eggs Unlike the situation in anurans, polyspermy is the rule in urodeles. While most studies have dealt with newts (suborder Salamandroidea), polyspermy has also been found in Cryptobranchus alleganiensis (Smith, 1912) and in Ambystoma mexicanum (SlBdeEek and Lanzovi, 1959; Wakimoto, 1979). Thus, representatives of three of the four suborders have physiological polyspermy (Table I). It is worth noting, however, that fertilization was usually monospermic in Hynobius retardatus (Makino, 1934). This finding should be confirmed and extended, since the family Hynobiidae is the most primitive family in the order, and this family may exhibit the ancestral mechanism of fertilization. In this section, the entry and fate of the sperm in urodele eggs will be described. A. SPERMENTRY

Insemination of the eggs of most urodeles is internal (see Salthe and Mecham, 1974). The male deposits a spermatophore bearing the sperm, which the female picks up with her cloaca. The sperm are stored in the spermatheca, and the eggs are inseminated as they pass through the cloaca to the outside. It is likely that the number of sperm per egg is regulated by this method of insemination. Like anuran eggs, urodele eggs are covered by several jelly layers secreted by the oviduct (Salthe, 1963), and sperm do not fertilize jellyless eggs (Good and Daniel, 1943; Nadamitsu, 1957; McLaughlin and Humphries, 1978; Matsuda and Onitake, 1984b). Matsuda and Onitake (1984b) have recently succeeded in fertilizing jellyless eggs of Cynops pyrrhogaster using sperm prepared in a high salt solution. This result suggests that, as with B . bufojaponicus (Ishihara et al., 1984), an important role of the jelly is to provide a proper ionic environment for fertilization. Sperm penetration through the jelly coats is rapid, and probably does not depend on the acrosome reaction. The acrosome reaction occurred only after the sperm had traversed the outer and middle jelly layers in Pleurodeles wultl, the only case investigated (Picheral, 1977a). On the basis of appearance of sperm entry sites, Fankhauser (1934a) estimated that in Triturus palmatus, most sperm entries into the egg occurred between 5 and 10 minutes after insemination. In Notophthalmus viridescens, many eggs had sperm in the cytoplasm within 5 minutes of insemination (Fankhauser and Moore, 1941a; McLaughlin and Humphries, 1978), and in P . wultl, sperm were at the surface of the egg within 3 minutes after insemination (Picheral, 1977b; Charbonneau et a l . , 1983b). As in

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anurans, the jelly hydrates when exposed to water, and the hydration places an absolute limit on the time available for sperm to fertilize the eggs. Jellied eggs of N . viridescens or C . pyrrhogaster became unfertilizable within 15 minutes of placement in water (McLaughlin and Humphries, 1978; Matsuda and Onitake, 1984a). The fact that most sperm entries occur in a limited time excludes the hypothesis that the relative time of entry is important in selecting sperm nuclei within these polyspermic eggs. Hydration causes not only the swelling of the jelly, as in anurans, but also the formation of a fluid-filled capsular chamber from the innermost jelly layer (Salthe, 1963; Picheral, 1977a; McLaughlin and Humphries, 1978). The formation of the capsular chamber plays a similar functional role to the perivitelline space of the anuran egg, and it may form in an analogous way. The perivitelline space in anurans forms between the egg and the vitelline envelope as the latter becomes the fertilization envelope. The fluid-filled perivitelline space results from cortical granule exocytosis-which produces an osmotic barrier, the F layer (Section I1,C)-and the influx of water allows the egg to rotate animal pole up. The urodele egg lacks cortical granules (Section III,B), and the mode of formation of the perivitelline space is not clear. With the capsular chamber, the egg in its vitelline envelope is free to rotate within the rest of the jelly so that the animal pole is up. An osmotic barrier must develop in the urodele jelly to allow water to be retained in the capsular chamber, and a candidate for the barrier is a lectin-ligand interaction between secretions of the anterior and middle oviducal regions (Jego et al., 1976, 1983a,b). Jego and co-workers consider that this interaction may be analogous to the cortical granule lectin-ligand interaction which produces the F layer in X . laevis. As mentioned, fertilization of urodele eggs is characterized by physiological polyspermy. More than one sperm normally enters the egg cytoplasm, and sperm entry occurs in both the animal and vegetal halves of the egg (van Bambeke, 1870; Jordan, 1893; Fankhauser and Moore, 1941a; Labrousse, 1966). The number of sperm which enter the egg is usually low, with modes of four sperm per egg in N . viridescens (Fankhauser and Moore, 1941a), four in C. pyrrhogaster (Streett, 1940), and three in T . palmatus (Fankhauser, 1932), and normal ranges of up to 15 in a variety of species (Smith, 1912; Fankhauser, 1932; Kaylor, 1937; Streett, 1940; Fankhauser and Moore, 1941a; Sladei-ek and Lanzova, 1959; Picheral, 1977b; McLaughlin and Humphries, 1978; Wakimoto, 1979; Charbonneau et al., 1983b; Iwao et al., 1985). Greater than 10 sperm per egg usually causes abnormal cleavage and development in T . palmatus (Fankhauser, 1932), while the N . viridescens egg cannot tolerate more than 13 (Kaylor, 1937). Entry of the sperm causes a local reaction, including the loss of microvilli at the surface and an accumulation of cytoplasm devoid of organelles inside (Picheral, 1977b). Later, each sperm entry site is recognized by a small depres-

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sion or pigment accumulation known as the sperm pit (Smith, 1912; Fankhauser, 1932; Fankhauser and Moore, 1941a; Charbonneau et al., 1983b). When eggs of the anuran X . laevis were inseminated with sperm of urodeles, sperm entered both the animal and vegetal halves (Jaffe et al., 1983; Iwao, 1985). The reaction zone surrounding the site of entry in this case was large, suggesting a zone of cytolysis. It is worth noting in this cross-order fertilization that the urodele sperm triggered the membrane depolarization as well as cortical granule exocytosis in the anuran egg. B. ACTIVATION OF THE EGG There are practically no reports of early events associated with egg activation in urodeles. There is little change in membrane potential either upon sperm entry or upon artificial activation in P. waltl and A . mexicanum (Charbonneau et al., 1983b), while inseminated eggs of C . pyrrhogaster exhibited several small, short-lived hyperpolarizations (Iwao, 1985). These results, along with the lack of evidence for Ca2 -activated chloride channels (Baud and Barish, 1984), suggest that there is no fast, electrical block to polyspermy. The absence of an electrical block was demonstrated by the entry of P . waltl sperm into eggs clamped at a positive membrane potential (Charbonneau et a l . , 1983b), the entry of N . viridescens sperm into anuran eggs either clamped at a positive potential or undergoing the normal fertilization potential (Jaffe et al., 1983), and the polyspermic entry of C . pyrrhogaster sperm into X . laevis eggs (Iwao, 1985). Therefore, not only do urodele eggs fail to exhibit a fertilization potential, but urodele sperm fail to respond to differences in membrane potential. Urodele eggs also lack a block to polyspermy mediated by cortical granule exocytosis, since cortical granules have not been found in T . alpestris, P . waltl, and N . viridescens (Wartenberg and Schmidt, 1961; Hope et al., 1963; Picheral, 1977b). Wartenberg (1 962) did not find cortical granules in A . mexicanum, although Ginzburg (1 97 1) claimed that oviducal eggs had cortical granules. It would be worthwhile to recheck eggs of A . mexicanum and to examine eggs of other species for cortical granules. There are no reports on changes in microvilli or pigmentation in the first few minutes of activation. All of the signs used to recognize activation of urodele eggs are scored about 1 hour after activation. These include a change of pigmentation from a rough, irregular appearance to a smooth, velvety appearance, the release of the second polar body, and the presence of even pigmentation over the animal half where previously the area around the animal pole lacked pigment (Signoret and Fagnier, 1962; Signoret et al., 1962; Charbonneau et al., 1983b). First, cleavage of fertilized eggs of many urodele species occurs about 7 hours after fertilization at 18°C (Bataillon, 1927; Kaylor, 1937; Stauffer, 1945; Labrousse, 1959), much later than the 1.5-3.5 hours characteristic of anuran eggs. Nonetheless, the relative timing of events, e.g., release of the second polar body, contact of the +

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female pronucleus with a male pronucleus, and mitosis, is similar to that of anuran eggs (Table 11). The urodele egg can be activated naturally by sperm and artificially by a heat shock (Signoret et al., 1962) or electrical shock (Signoret and Fagnier, 1962). Pricking the egg with a needle, a procedure which works well for anuran eggs, generally does not activate a urodele egg (Lehman, 1955; Signoret et al., 1962), although eggs of P . waltl have been successfully prick-activated (Labrousse, 1971a; Aimar and Labrousse, 1975). Activation of P . waltl eggs with A23187 has been reported recently (Charbonneau et al., 1983b). There is an increase in the level of the Ca2 -regulatory protein, calmodulin, after fertilization in P . waltl (Gallien et al., 1984), but the relationship between this rise and the events of activation is unknown. A wave of free Ca2 appears to be involved in the activation of eggs of many vertebrates, and Jaffe (1983) has argued that this may be a universal feature in the activation of all deuterostome eggs. As seen from the information presented above, however, there is presently little evidence that a wave of changes is initiated from the site of sperm entry in urodele eggs or that calcium is involved in the activation of the egg. Given the quantity of evidence that Jaffe has marshalled for his hypothesis, it would be worth examining activation of urodele eggs carefully to see whether or not it is similar to activation of eggs of other vertebrates. +

+

C. DEVELOPMENT OF SPERMAND EGG NUCLEI The cytological events following sperm entry have been described in C . alleganiensis (Smith, 1912), T . alpestris and T . cristatus (Bataillon and Tchou Su, 1929, 1930), T . palmatus (Fankhauser, 1932), H . retardatus (Makino, 1934), N . viridescens (Fankhauser and Moore, 1941a), P . waltl (Labrousse, 1959, 1966, 1971a,b), A . mexicanum (SlBdeEek and LanzovB, 1959; Wakimoto, 1979), and C . pyrrhogaster (Iwao et al., 1985). During the first half of the first cell cycle following fertilization, the sperm nuclei all behave similarly, with the exception that nuclei in the more yolky vegetal-half lag behind those in the less yolky animal-half cytoplasm. The nuclei transform from very elongated sperm nuclei with condensed chromatin to enlarged nuclei with dispersed chromatin (Bataillon and Tchou Su, 1929; Fankhauser, 1932; Fankhauser and Moore, 1941a; Labrousse, 1971a,b; Wakimoto, 1979). All of the sperm nuclei begin to synthesize DNA along with the egg nucleus. DNA synthesis begins before the egg nucleus associates with a sperm nucleus and continues after their contact (Labrousse, 1971a,b; Wakimoto, 1979; Iwao et al., 1985). Each sperm nucleus is surrounded by a growing sperm-aster, which, as in anurans, does not fuse with other spermasters and keeps the nuclei apart (Fankhauser, 1932; Fankhauser and Moore, 1941a). Contact between the egg nucleus and a sperm nucleus (now designated the

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principal sperm nucleus) occurs midway through the first cell cycle (Table 11). Prior to the entrance of the egg nucleus into the aster of a sperm nucleus, it is not possible to decide which sperm nucleus will be the principal one and which will become accessory nuclei (Fankhauser and Moore, 1941a). As in anurans (Subtelny and Bradt, 1963; Ubbels et al., 1983), the large sperm-asters fade as the nuclei prepare to enter mitosis (Fankhauser, 1932; Fankhauser and Moore, 1941a). Shortly after nuclear contact, the first differences appear between the principal sperm nucleus and the accessory ones. The chromatin in the accessory nuclei condenses on the side of the nucleus which is in contact with the centrosome. The distinct centrosome is often located in a pocket of the nuclear surface and has a few astral rays extending from it (Fankhauser, 1932; Fankhauser and Moore, 1941a; Wakimoto, 1979). Two small asters are associated with the principal sperm nucleus and the egg nucleus, and those become the poles of the first cleavage spindle. In contrast, a monaster is usually associated with the accessory nuclei, indicating the presence of an unreplicated or unseparated centrosome (Fankhauser, 1932; Fankhauser and Moore, 1941a). The subsequent degeneration of the accessory nuclei has been described differently by different authors. Bataillon and Tchou Su (1929) reported that in T . alpestris, some accessory nuclei develop as far as prophase and may even have a divided aster, while other accessory nuclei are less advanced. All of the accessory nuclei degenerate by the time the zygote nucleus is in telophase. Fankhauser (1932) found in T . palrnatus that the accessory nuclei formed groups of vesicles or condensed chromatin which persisted during cleavage of the embryo. Finally, Fankhauser and Moore (1941a) saw that the accessory nuclei frequently formed prometaphase and other mitotic figures at the time of first cleavage, although their appearance was delayed relative to the zygote nucleus. They suggested that these delayed mitotic states may have been missed in earlier studies. As the egg begins to cleave (Fig. 3), the accessory nuclei are usually found at the periphery of the egg or in the vegetal region (Bataillon and Tchou Su, 1930; Fankhauser, 1932; Fankhauser and Moore, 1941a). Since the male pronuclei had originally been located throughout the animal cytoplasm, Fankhauser (1932; also, Fankhauser and Moore, 1941a) suggested that they were pushed out of the central animal region by the large asters of the first mitotic spindle.

IV. Control of Accessory Sperm Nuclei in Urodele Fertilization A. THE HYPOTHESES OF BATAILLON AND FANKHAUSER Two hypotheses have been proposed to account for the degeneration of the accessory nuclei. Bataillon and Tchou Su (1930) suggested that the development

81

FERTILIZATION IN AMPHIBIANS Bataillon's Hypothesis

Fankhauser's Hypothesis

0.3

0.5

0.9

FIG.4. Bataillon's and Fankhauser's hypotheses. The hypotheses are compared at three times in the first cell cycle, indicated by the normalized times 0.3,0.5, and 0.9. With Bataillon's hypothesis, one sperm nucleus enters an area of active cytoplasm (stippled), where it encounters the female nucleus. The active cytoplasm allows the formation of a bipolar spindle and other cell cycle events. Sperm nuclei not stimulated by the active cytoplasm degenerate. With Fankhauser's hypothesis, an activator (A) from the female nucleus attracts a male nucleus. The combined male and female nuclei then release an inhibitor (I) which prevents the normal development of the accessory nuclei and they degenerate.

of each sperm nucleus and its aster depended on the amount of active cytoplasm available to it (Fig. 4). The active cytoplasm is limited in quantity and is concentrated in the center of the animal half. The sperm nucleus which occupies the center becomes the principal nucleus, and due to the position and the large sperm-aster of this nucleus, the egg nucleus approaches and contacts it, thus forming the zygote nucleus. The zygote nucleus proceeds into mitosis, while all accessory nuclei lag by various degrees since they are in poorer cytoplasmic regions. As the dominant zygote nucleus enters mitosis, the metabolism of the egg which is linked to this mitosis changes. The incompatibility between the lagging accessory nuclei and the new conditions in the egg leads to the degeneration of the accessory nuclei. To test this hypothesis, Bataillon (1927) attempted to provide active cytoplasm to more than one sperm nucleus with the expectation that synchronous mitosis and multiple cleavage furrows would be produced. He lightly centrifuged eggs of T . alpeshis at the time of fertilization in order to concentrate more active cytoplasm in the animal half, and obtained multiple furrows in one third of the

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cases. Preliminary cytological evidence suggested that more than one sperm nucleus underwent mitosis in accordance with Bataillon’s hypothesis. In contrast, Fankhauser (1925, 1932, 1934a, 1948), following an idea of Spemann (1914), proposed that there are inhibitors and activators which come from certain nuclei which influence the behavior of other nuclei (Fig. 4). He suggested that an activator emanates from the egg nucleus which enhances the development of one sperm nucleus and its aster, and in this way, one of the sperm nuclei is chosen for further development. Fankhauser proposed further that an inhibitor diffused from the contacting sperm and egg nuclei, which caused the degeneration of the accessory nuclei. Fankhauser’s hypothesis was based on a series of experiments in which he used a hairloop to separate the fertilized egg into a fragment containing the egg nucleus as well as sperm nuclei and a fragment with only sperm nuclei (Fankhauser, 1925). The fragments containing only sperm nuclei frequently divided, implying that the sperm in the absence of the egg nucleus and its associated sperm nucleus had not been inhibited and were able to undergo mitosis. He also partially constricted the egg, leaving a channel between the half with the egg nucleus and the half with only sperm nuclei. When the channel was small, the half with the sperm nuclei cleaved but when the channel was large, that half did not cleave. Fankhauser’s interpretation of these results was that the inhibition was chemical and by constricting the egg sufficiently the inhibitor could not reach the sperm nuclei in the other half. Bataillon and Tchou Su (1930) felt that Fankhauser’s explanation was unnecessarily complex and urged him to examine the cytology of the eggs in detail. Fankhauser accepted the challenge and spent more than a decade in this task. He found that the behavior of the sperm nuclei was quite variable and judged that the suppression of the accessory nuclei was much more complicated than he had previously thought (Fankhauser, 1934a,b; Fankhauser and Moore, 1941b).

B . ANDROGENETIC AND GYNOGENETIC DEVELOPMENT IN URODELES Bataillon and Tchou Su argued that one sperm nucleus developed in advance of others due to the stimulatory effect of the area of cytoplasm in which it found itself. In contrast, Fankhauser hypothesized that the female nucleus had a stimulatory effect on one sperm nucleus and then the associated sperm-egg nuclei had an inhibitory effect on the accessory sperm nuclei (Fig. 4). Theoretically, it should be possible to distinguish between these hypotheses by observing androgenetic development; that is, development following removal of the female nucleus. According to Bataillon, the removal should have no effect, while according to Fankhauser, the removal should yield the continued but slower development of many sperm nuclei. Unfortunately, this simple test has not yielded a simple answer, in part due to a possibility suggested by Morgan (1927). Morgan

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83

commented that a stimulation of development of one sperm nucleus need not be due to the female nucleus but to the cytoplasm immediately surrounding it. Not only does this proposal blur the distinction between Bataillon’s and Fankhauser’s hypotheses, but as will be seen, it makes ambiguous the interpretation of most of the experiments on androgenesis. Androgenesis was produced in two ways. The first method was to cut the egg into two pieces, one of which contained only sperm nuclei (andromerogonic fragment), and the second method was to suck the female nucleus out of the egg using a pipette. The results of these experiments supported Fankhauser in that the androgenetic eggs or fragments had multiple furrows indicative of activity of several sperm nuclei and the furrows were delayed in forming (Table 111). Very few eggs had a normal furrow, indicative of a bipolar spindle, and development was poor, indicating that only in a few cases did one sperm nucleus become dominant. Egg fragments containing the egg nucleus as well as sperm nuclei cleaved and developed normally (Fankhauser, 1925, 1930). It should be noted that haploid androgenetic development is routinely achieved in anurans by fertilizing eggs and removing the egg nucleus (Porter, 1939; Briggs, 1946; Gurdon, 1960). The experiments on urodele androgenesis are open to criticism on several grounds. It is not clear whether the abnormal, delayed furrowing seen in the androgenetic eggs was due to the independent, uninhibited activity of sperm nuclei, or whether the furrowing was more of the abortive type such as that found with activated eggs. If the latter is true, the androgenetic eggs may appear to have better “cleavage” than activated eggs due to the presence of several sperm centrosomes. Cytological examination showed that the sperm nuclei behaved heterogeneously, with many nuclei forming no aster or a monaster (Fankhauser, 1934a; Fankhauser and Moore, 1941b). There was no clear relationship between the behavior of the nuclei and the furrowing pattern, as is the case for polyspermic anuran eggs (Fankhauser, 1934b; Fankhauser and Moore, 1941b). Finally, these experiments fail to distinguish between Bataillon’s and Fankhauser’s hypotheses, since cytoplasm as well as the female nucleus was removed. This cytoplasm would have been near the female nucleus and in Morgan’s opinion would be potentially important for sperm nuclear behavior. The significance of the cytoplasmic loss is illustrated by fragmentary evidence from nuclear transplantation studies. In nuclear transplants, a diploid nucleus complete with its centrosomes is placed into an activated egg following removal of the female nucleus. While this procedure is very successful in producing embryos with anurans (McKinnell, 1978), abnormal furrows and no development occurred when initially tried with urodeles (Waddington and Pantelouris, 1953; Lehman, 1955). In these early attempts, the female nucleus was removed by cutting of the egg or by suction, and it was not until Signoret et al. (1962) inactivated the female nucleus with ultraviolet (UV) light that success was ob-

TABLE I11 ANDROCENETIC DEVELOPMENT IN URODELES Cleavage

Species

Experiment

Development Beyond gastrulation

No. cleaved

Normal

(%)

(%)

Comments Furrow delayed by 1-2 hours; fragments with egg nucleus had 68% normal cleavage Furrow delayed by 0.5-1.5 cycles Furrow delayed

T. palmatus (Fankhauser, 1925)

Andromerogonic fragment

89 (97)

12

T . palmatus (Fankhauser, 1943b) N . viridescens (Kaylor, 1937) A. mexicanurn (Stauffer, 1945) T. palmatus, T. alpestris (Lehman, 1955) C. pyrrhogaster (Iwao et a l . , 1985)

Andromerogonic fragment

259 (93)

9

Androgenesis by suction

128 (89)

25

Androgenesis by suction

103 (14)

36

Nuclear transplantation; enucleated by suction

163 (65)

34

Most with normal furrows had a 3-hour delay Furrow delay of 0.5-3 hours

51

Furrow delay of 1-1.5 hours

0

Multipolar divisions delayed by 1-2 hours 40 Normal blastulae from 60

A. mexicanurn (Signoret et al., 1962) A. mexicanurn (Brothers, 1979) A. mexicanurn (J. B. Armstrong 1984, personal communication)

Andromerogonic fragment Androgenesis by suction UV androgenesis

-

67

UV androgenesis

143 (93)

72

UV androgenesis

308

89

eggs

-

No. raised

(%)

-

-

154

12

128

11

-

-

36

0

-

40

98

94

81

215

83

FERTILIZATION IN AMPHIBIANS

85

tained. There are several reasons for the success since several changes in protocol were made, but one important change may be that the female nucleus was inactivated without removal of cytoplasm. Since then, normal early development of androgenetic haploids has been obtained in A. mexicanum by inseminating eggs whose nuclei were UV inactivated (Table 111). In light of Bataillon’s and Fankhauser’s hypotheses, it would be worth asking what the frequency is of normal first cleavage and whether cleavage is delayed or not in the UVenucleated eggs. The development of gynogenetic haploids provides further information on the nature of the suppression of the accessory nuclei. Gynogenetic haploids, developing with only the female nucleus, are produced by fertilizing eggs with sperm whose nuclei have been genetically inactivated with UV. Selman (1958) demonstrated the feasibility of this procedure on several Triturus species, and this was followed by a careful study on A. mexicanum by Hronowski et al. (1979). With A. mexicanum, there were no differences between diploid and gynogenetic haploid embryos with respect to the frequency of normal cleavage or the time of first cleavage. Assuming the eggs were polyspermic, these results suggest that the sperm nucleus is neither the source nor the target of an inhibitor. The principal sperm nucleus is not the source of suppression of accessory nuclei in these eggs since the principal sperm nucleus would be genetically inactive. The accessory nuclei are not the target of suppression since they are already inactive. Rather, the normality of cleavage suggests that the centrosomes associated with the accessory nuclei fail to function in the gynogenetic eggs, and in this respect, the situation is the same as in normal diploid development. C. A NEW LOOKAT

THE

OLDHYPOTHESES

While little has been done regarding physiological polyspermy in the last 40 years, time has been kinder to Bataillon than to Fankhauser. The idea that the cytoplasm influences the nucleus, particularly with respect to nuclear behavior in the cell cycle, is a well-established tenet of cell biology (Johnson and Rao, 1971; Masui and Clarke, 1979; Newport and Kirschner, 1984). On the other hand, the idea of activators and inhibitors, emanating from nuclei, has little current support. The egg nucleus does exhibit genetic activity early in the first cell cycle in A. mexicanum (Signoret et al., 1981; Lefresne et al., 1983), but inhibition of transcription by a-amanitan in C. pyrrhogaster did not prevent the normal suppression of the accessory nuclei (Iwao et a f . , 1985). One difficulty in opposing Fankhauser’s hypothesis, however, is how to explain the results of his constriction experiments (Section IV,A), recently repeated by Iwao et al. (1985). In these experiments, the ability of accessory nuclei to promote cleavage was inversely related to the size of the channel separating the half of the egg with accessory nuclei and the half of the egg with a male and

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RICHARD P. ELINSON

female nucleus. This experiment is the cornerstone of Fankhauser’s hypothesis of a diffusible inhibitor which suppresses the accessory nuclei. A.A. Humphries (personal communication; Barbieri and Humphries, unpublished information) has suggested a reinterpretation of these results. He proposes that the tighter the constriction, the more likely that accessory nuclei will be brought into contact with active cytoplasm. As a result, the development of these nuclei will be enhanced. Humphries has further suggested that the source of active cytoplasm is the germinal vesicle, the large nucleus of the oocyte. It has long been known that the contents of the germinal vesicle are necessary for cell division of the fertilized egg (Wilson, 1925; Smith and Ecker, 1969), and recent work has demonstrated that germinal vesicle contents are required for decondensation of the sperm nucleus and DNA synthesis (Katagiri and Moriya, 1976; Lohka and Masui, 1983). The accessory nuclei in urodele fertilization decondense and synthesize DNA, so this does not seem to be the cause of their lack of development. Rather, the most obvious difference between the accessory nuclei and the fusion nuclei is the failure of the former to make a bipolar spindle. Work on frog and starfish eggs has shown that germinal vesicle contents are not necessary for the formation of a sperm aster (Katagiri and Moriya, 1976; Hirai et al., 1981), although they are necessary for cleavage. This raises the possibility that a component from the germinal vesicle is involved in centrosome replication or separation and that the accessory nuclei in urodele eggs form only a monaster due to the restricted distribution of this hypothetical component. It is interesting to note that Imoh and Miyazaki (1984) have reported that the germinal vesicle contents are more restricted in their distribution in the oocyte of the urodele C . pyrrhogaster compared to the anuran X . laevis. Another hypothesis, which currently cannot be rejected, is that the bipolar spindle of the first cleavage division in the urodele egg requires an egg nuclear component and/or an associated centrosome. This is certainly not the case in many marine invertebrates and in anurans. With eggs of these animals, the organization of the spindle depends on the sperm as demonstrated by the normal cleavage of androgenetic fragments, the failure of cleavage in artificially activated eggs, and the multiple cleavage furrows in polyspermic eggs (Wilson, 1925; Fig. 3). As described, urodele eggs behave differently with respect to androgenesis and polyspermy . The successes at androgenesis in urodeles (Table 111) have been accomplished using UV inactivation of the female nucleus, thus leaving in the egg nuclear components which may be used for spindle formation. The possibility that the urodele egg contributes a centrosome would appear to be unlikely, however, since the egg nucleus in fertilized or artificially activated eggs usually does not have an aster associated with it (Fankhauser, 1932, 1948; Fankhauser and Moore, 1941a; Labrousse, 1959; Aimar and Labrousse, 1975). Nonetheless, all of the experiments on urodele eggs to date can be explained by the hypothesis that an egg nuclear component and/or an associated centrosome is required for a bipolar spindle and normal cleavage.

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87

The above considerations suggest several experimental approaches to the problem of the control of accessory nuclear activity. The restricted movement of germinal vesicle contents in the urodele egg as indicated by cytochemical tests (Imoh and Miyazaki, 1984) can be explored further using monoclonal antibodies togerminal vesicle proteins (Dreyer e t a l . , 1982).The expectation is that there will be clear differences between eggs of anurans and urodeles in the distribution of common molecules. Whether or not the germinal vesicle is important, it should be possible to change the arrangement of cytoplasm in a urodele egg and to see whether this affects the development of accessory nuclei. Bataillon (1927) used centrifugation to enhance development of accessory nuclei, and these results should be repeated with more complete cytological observations on the nuclei and on the arrangement of the cytoplasm. The arrangement of the cytoplasm probably depends on the cytoskeleton, so it should be possible to affect the arrangement by altering the cytoskeleton. Beetschen ( 1958) observed development of accessory nuclei in P . waltl when fertilized eggs were subjected to long periods in the cold. The cold may be acting through the cytoskeleton, since in X . laevis, cold can affect both the cytoplasmic arrangement and the cytoskeleton (Scharf and Gerhart, 1983; Elinson, 1985). Another way to affect cytoplasmic arrangement is to transfer cytoplasm or nucleoplasm from the germinal vesicle between eggs by microinjection. Again, the objective is to see whether accessory nuclei can be rescued by selected cytoplasmic components. Finally, it would be valuable to follow centrosome replication in urodele eggs, perhaps by adapting methods for immunological detection of centrosomes (Calarco-Gillam et al., 1983) to the large amphibian egg. Such a method could test whether the female nucleus has an associated centrosome and could determine what happens to the centrosome of an accessory nucleus. V. The Ancestral Egg A. COMPARISON BETWEEN ANURAN AND URODELEFERTILIZATION

A comparison between fertilization in anurans and urodeles shows a mirrorimage dichotomy with respect to polyspermy (Table IV). The anuran egg has a fast and slow block to polyspermy, and it has no protection when more than one sperm enters. The urodele egg lacks both external blocks to polyspermy but can deal with accessory nuclei within the egg. From the experiments discussed previously, it is possible to draw up a list of the minimal components required by the anuran and urodele eggs to ensure that only one sperm nucleus fuses with the egg nucleus. The anuran block to polyspermy requires elaborate machinery. The egg has Ca2 -sensitive chloride channels whose opening produces the membrane depolarization involved in the fast block to polyspermy. The egg also has Ca2++

88

RICHARD P. ELINSON TABLE IV COMPARISON U-ANUKAN A N D UKODELE G A M t T t S WITH

Egg Normal fertilization Site of sperm entry Fast block to polyspermy Fertilization potential Chloride channels Slow block to polyspermy Ca*+ wave Cortical granule exocytosis Alteration of vitelline envelope Development of supernumerary sperm nuclei in polyspermic eggs Sperm Can trigger membrane depolarization Can trigger cortical granule exocytosis Responds to egg membrane potential

RESPECTTO

POLYSPERMY

Anurdn

Urodele

Monospermic Animal half

Polyspermic Animal and vegetal halves -

+ + + +

Probably .t

+

+

+ + +

-

> -

No data No data -

+ + -

sensitive cortical granules which contain a lectin and hydrolases. The interaction of the lectin with a ligand secreted around the egg by the oviduct and the hydrolysis of molecules in the vitelline envelope produce the fertilization envelope, which serves as a slow block to polyspermy. The sperm, for its part, may have a voltage-sensitive molecule, allowing it to respond to the membrane depolarization of the egg. While these molecules and organelles are the minimal components used by the anurans to ensure monospermy, the list is already a long one. More than the length of the list, the arrangement of components in the membranes and in the cortical granules is important for the polyspermy-blocking reactions. The urodele block to polyspermy requires a cytoplasmic activity of unknown character or complexity which permits the development of only one sperm nucleus. The uncertainty of the nature of the cytoplasmic activity makes it difficult to speculate on the number of components required. This is especially true if a hypothesis like Fankhauser’s, with its novel nuclear activities, is correct. On the other hand, if a hypothesis like Bataillon’s is correct, then the lack of development of accessory nuclei would be simply due to the restricted distribution of a common cytoplasmic activity within the urodele egg. A minimal change in the arrangement of the cytoskeleton or in the association of the cytoplasmic activity with other components in the egg is all that would be required to change the urodele egg response to the anuran egg response. The accounting presented here gives an indication of the minimal cellular

FERTILIZATION IN AMPHIBIANS

89

differences between the mechanisms directed at polyspermy in the anuran and urodele eggs. The evolutionary question can be raised as to whether it is feasible to think of converting one egg type to the other. To approach this question, I will consider the situation in fish to see whether the mechanisms in these animals give us clues about the ancestral amphibian egg. Following that, I will consider two amphibian cases in which a conversion between the anuran and the urodele mechanisms may be occumng. B. BLOCKSTO POLYSPERMYIN FISH Attempting to determine the nature of the ancestral amphibian egg by looking at fertilization in fish is an approach fraught with difficulty. First, the group of fish from which the amphibians evolved has not been definitively established (Thomson, 1968; Rosen et al., 1981). Second, practically nothing is known about fertilization in the fishes which are candidates for the closest amphibian relatives. Finally and most importantly, the amphibians diverged from fishes about 400 million years ago. Obviously, the eggs of both amphibians and fish have had a tremendously long time to change since their last common ancestor. Nonetheless, the similarities seen in fish fertilization suggest that some aspects of the primitive pattern may have been conserved. Studies on fertilization in the bony fishes (class Osteichthyes) have concentrated on the chondrosteans and the teleosts of the subclass Actinopterygii. These animals all have monospermic fertilization (Ginzburg, 1968) and appear to use a similar mechanism to block polyspermy (Ginzburg, 1968; also see Gilkey, 198 1 , for reviews). The egg is surrounded by a tough extracellular coat called the chorion which has one or a few micropyles. The micropyle is a small hole which is just large enough to permit the passage of one sperm at a time through the chorion. The presence of a hole in the chorion obviates the need for the sperm to digest its way to the egg surface, and sperm of many teleosts lack acrosomes. The micropyle restricts the site of sperm entry to a tiny area of the egg surface, and removal of the chorion permits multiple entries at other locations (see Kobayashi and Yamamoto, 198 1 , for references). The egg has cortical granules or cortical alveoli which undergo exocytosis when the sperm enters the egg. The contents of the cortical granules block sperm passage through the micropyle, although a change in the plasma membrane at the site of sperm entry may play a role in preventing polyspermy (Brummet and Dumont, 198 1 ; Kobayashi and Yamamoto, 1981; Hart and Donovan, 1983; Ohta and Iwamatsu, 1983). The egg of one fish, the teleost Oryzias latipes, has been examined for an electrical fast block to polyspermy, and like the urodeles, it lacks one. There is only a small membrane depolarization upon sperm entry, and entry is not prevented when the membrane potential is artificially altered (Nuccitelli, 1980). While the above investigations give a consistent picture in the Actinopterygii,

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RICHARD P. ELINSON

there is little information on fertilization in the Sarcopterygii, the subclass of Osteichthyes containing the closer relatives to amphibians. The extant forms include several species of lungfish and the coelacanth Latimeria. It is not known whether the eggs of those animals have cortical granules or a chorion with a micropyle. Sperm of a lungfish (Boisson and Mattei, 1965; Jespersen, 1971) and of Latimeria (Tuzet and Millot, 1959, cited in Ginzburg, 1968) appear to have acrosomes, raising the possibility that the sperm do not enter the egg through a micropyle. C. HYPOTHETICALANCESTRIES It seems likely that the ancestral amphibian egg had cortical granules and other mechanisms to ensure monospermy. There are two reasons for this conclusion. First, Ginzburg ( I 968) has noted the widespread occurrence of monospermy and the presence of cortical granules in eggs of fish and many other animals. On this basis, Ginzburg argued that monospermic fertilization is the primitive type and that physiological polyspermy was derived from it. Second, the mechanisms to block polyspermy in the anurans are complex, and it is easier to imagine the loss of organelles and molecules involved in these mechanisms than to imagine their de now appearance. Therefore, in proposing ancestries, I will start with eggs which have some of the anuran mechanisms. Most anurans examined with respect to fertilization conform to the general patterns already discussed. There are, however, two rather different anuran egg types which may help to bridge the gap between anurans and urodeles. First, D. picrus exhibits many interesting differences in its fertilization pattern, the most striking being the restriction of sperm entry to a small point at the animal pole of the egg. Second, many anurans have very large eggs (greater than 3 mm in diameter) which are fertilized on land, in contrast to the small eggs (1.3-2 mm in diameter) fertilized in water which are usually studied. I will use the differences seen in D.pictus and the modifications due to large egg size to propose models for the transition between the anuran and urodele egg types. 1. The Discoglossus Model The D.pictus egg has an unusual jelly arrangement which restricts the site of sperm entry. In addition to the outer jelly layers which surround the egg completely, there is a plug of jelly, the animal plug, which indents the animal half of the egg. At the base of this animal depression, there is a small pit known as the animal dimple (Campanella, 1975). The sperm of D.pictus have acrosomes and are 2.3 mm long, one of the longest types of sperm known (Campanella and Gabbiani, 1979). The sperm penetrate the animal plug and are all directed toward

FERTILIZATION IN AMPHIBIANS

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the animal dimple where sperm-egg fusion occurs. Whether sperm can enter elsewhere on the surface has not been tested, but the egg surface at the animal dimple is different from the rest of the surface, as indicated by lectin binding (Denis-Donini and Campanella, 1977). Upon activation by sperm or a needle, the egg membrane depolarizes as in other anurans (Talevi er al., 1985). The D . pictus egg appears to have cortical granules, but they are unusual in several respects. They are smaller than those in other anurans, they lie somewhat removed from the cell membrane, and most importantly, they are more concentrated in the area around the animal dimple (Campanella, 1975). The rest of the peripheral cytoplasm lacks these granules. The granules are Golgi derived, as expected (Andreuccetti and Campanella, 1980), and they disappear with fertilization (Denis-Donini and Campanella, 1977). It is not known whether the membrane depolarization or the cortical granules are involved in a block to polyspermy in D . picrus. The lack of cortical granules around the egg is correlated with the fact that a capsular chamber forms which allows egg rotation as in urodeles. The capsular chamber forms by liquefaction of the animal plug, and this is due primarily to a secretion from the egg (Hibbard, 1928; Wintrebert, 1929, 1933). This mode of formation is different from urodeles in which the capsular chamber forms by hydration alone. The unusual fertilization mechanisms in D . pictus may have been derived from the typical anuran pattern, or conversely, they may represent the primitive condition. The latter possibility is made plausible by the phylogeny of D. pictus. D . pictus is a member of the family Discoglossidae, which is considered one of the oldest anuran families on the basis of anatomy and the fossil record (Estes and Reig, 1973; Lynch, 1973). The only family considered more ancient is the Leiopelmatidae, which is included with Discoglossidae in the superfamily Discoglossoidea (Duellman, 1975; Laurent, 1979). Nothing is known about fertilization mechanisms in the Leiopelmatidae, so they cannot be used to judge whether fertilization in D. picrus follows the primitive pattern. Within the family Discoglossidae, there are three genera, Discoglossus, Alytes, and Bombina. Alyres obsretricans, whose lineage diverged from Discoglossus about 90 million years ago (Maxson and Szymura, 19841, has a capsular chamber (Salthe, 1963), raising the possibility that these eggs also do not have cortical granules around the whole egg. On the other hand, Bombina orientalis, whose lineage diverged about 70 million years ago (Maxson and Szymura, 1984), has cortical granules around the whole egg surface, and sperm entry is not restricted to the animal pole (Pasceri and Elinson, unpublished information). The Bombina result places an outside limit on the time required for the transition between the D . pictus egg and the typical anuran egg. If D. pictus is indeed primitive in terms of its fertilization mechanism, then the typical cortical granule-mediated, monospermic anuran egg

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RICHARD P. ELINSON

type may have evolved twice, once in the more modem anurans sometime after their divergence from the superfamily Discoglossoidea and once in Bombina after its divergence from Discoglossus. I will assume, based on D . pictus, that the primitive amphibian egg restricted the site of sperm entry to the animal pole due to the extracellular coats, and had components which could be utilized for a block to polyspermy near this site. Furthermore, I will assume that the cytoplasmic components required for pronuclear development and other cell cycle events were localized near the animal pole where the sperm nucleus would be. To obtain the typical anuran pattern of fertilization, the polysperrny block mechanisms would gradually occupy more of the egg surface, allowing a concomitant reduction in the restrictions on sperm entry site by the extracellular coats. To obtain the typical urodele pattern of fertilization, the sperm would lose their sensitivity to the membrane potential and the animal-vegetal differences of the egg plasma membrane. A loss of the restrictions imposed by the extracellular coats on the site of sperm entry would leave the egg surface unprotected against polyspermy. Most of the sperm nuclei would not develop normally, however, since they would not be exposed to the required cytoplasmic components localized near the former site of sperm entry at the animal pole. While this model does not include many details, it illustrates the feasibility of deriving the anuran egg and the urodele egg from a common ancestral egg. There are several experimental tests of this scenario using D . pictus. First, it would be useful to know whether the entry of D . pictus sperm is affected by the membrane potential of the egg. Although a fertilization potential exists in D . pictus (Talevi et a l . , 1985), its role as a fast block to polyspermy in this species has not been determined. Second, it should be possible to determine whether certain cytoplasmic components such as the germinal vesicle contents show a diffuse distribution or are restricted to the animal pole area. The latter possibility is suggested by cytochemical studies (Wintrebert, 1933; Klag and Ubbels, 1975). Finally, it would be interesting to see the fate of sperm nuclei when entry is not at the animal dimple. By interspecies transfer of jellyless eggs (Elinson, 1974b), D . pictus eggs could be wrapped by Bombina jelly and inseminated. Under these conditions, sperm entry may not be restricted to the animal pole, and polyspermy may occur. 2 . The Large Egg Model While most anuran eggs are less than 2 mm in diameter, some species produce much larger eggs (Salthe and Duellman, 1973; del Pino and Escobar, 1981). The large eggs are adaptations for terrestrial development, and their large size is due to the huge increase in the amount of yolk required for development on land to an advanced tadpole or to a frog stage. Sperm which enter yolky areas of the small, aquatic R . pipiens eggs have poor nuclear development and usually do not induce

FERTILIZATION IN AMPHIBIANS

93

vegetal cleavages (Elinson, 1977), so it might be expected that very large, yolky eggs could tolerate multiple sperm entry into the yolk with no adverse developmental consequences. Cytoplasmic conditions which permit cell cycle events could be restricted to the yolk-poor areas around the animal pole since that is where the initial cell divisions occur. Given this situation, the polyspermy block mechanisms over most of the egg surface could be relaxed without deleterious effect. As a result, this anuran egg approaches the urodele case by permitting supernumerary sperm entry and restricting development of sperm nuclei to a small region of the cytoplasm. The many anuran lineages which have species with large eggs permit observations which may demonstrate the reality of the above sequence. The large eggs should be examined for the presence of cortical granules all the way around the egg, the ability of the egg to respond to local activation such as a needle prick in different areas, and the presence of supernumerary sperm in the yolky areas.

VI. Conclusion Fertilization in anurans and urodeles is so different that it represents an important character distinguishing the orders. After all, fertilization is the event which initiates development, so basic changes in its mechanisms, which must have occurred in amphibian evolution, are not expected. It should be possible to examine what is involved in changing one pattern of fertilization to another both by experiments and by observations, and I have suggested several approaches for analyzing physiological polyspermy in urodeles (Section IV ,C) and for finding routes of conversion between the urodele and anuran egg responses (Section V,C). While these experimental approaches would define better what is possible, the evolutionary question is not how easy it is to overcome the anuran-urodele fertilization difference experimentally, but how this difference arose. Here it should be noted that while investigators have looked at fertilization in the major groups of anurans and urodeles, the most primitive families in each order have not been extensively studied. This is particularly important since most of the variation in amphibian fertilization has been found in the primitive families. Among anurans, Ascaphus truei of the family Leiopelmatidae has internal fertilization, and as discussed, D. pictus has greatly modified fertilization. Among urodeles, the most primitive families, Hynobiidae and Cryptobranchidae, have external fertilization, and H . returdatus may be monospermic. These deviations from the patterns characteristic of their orders suggest that it would be fruitful to examine fertilization in the anuran families Leiopelmatidae and Discoglossidae (superfamily Discoglossoidea) and in the urodele families Hynobiidae and Cryp-

94

RICHARD P. ELINSON

tobranchidae (suborder Cryptobranchoidea). Such observations would help to clarify the anuran-urodele differences in fertilization. Amphibians have long been favorite experimental animals for the study of fertilization and embryogenesis because they provide the researcher with easy access to a large number of eggs. Besides this advantage, the amphibians also exhibit a great diversity of reproductive patterns (Salthe and Duellman, 1973; Salthe and Mecham, 1974; Lamotte and Lescure, 1977). This diversity should make the amphibians ideal animals for examining the cellular evolution of developmental processes.

ACKNOWLEDGMENTS I thank R. D. Grey, D. M. Green, A. A . Humphries, Jr., and T. S. Parsons for critical comments on this manuscript. I thank C. Aimar, J. B. Armstrong, A. J. Brothers, C. Campanella, J . Hanken, A. A. Humphries, Jr., C. Katagiri, G . M. Malacinski, and R. Nuccitelli for useful discussions or for supplying unpublished information. The writing of this review was supported by Grant A6356 from Natural Sciences and Engineering Research Council of Canada.

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