Structure and function of the extracellular matrix of anuran eggs.

JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 17:319-335 (1991)

Structure and Function of the Extracellular Matrix of Anuran Eggs JERRY L. HEDRICK AND TATSURO NISHIHARA Department of Biochemistry and Biophysics, University of California, Dauis, California 9561 6

KEY WORDS

Envelope, Jelly coat, Fertilization, Sperm, Lectin, Glycoprotein, Protease

ABSTRACT

The extracellular matrix (ECM) surrounding the anuran egg is composed of jelly coat layers, an envelope, and the perivitelline space, which separates the envelope from the egg plasma membrane. Both the jelly coat layers and egg envelopes are required for fertilization in anurans. This paper reviews the current understanding of the structure-function relations of the ECM, with emphasis on the egg envelope. The fibrous egg envelope exists in four related forms. The envelope forms differ in their ultrastructures, macromolecular compositions, and cellular functions. After the oocyte is released from the ovary, conversion of one envelope form to another is brought about by factors secreted by the oviduct prior to fertilization and by factors released from the egg in the sperm-triggered cortical reaction. An additional extracellular matrix structure, located in the perivitelline space, has recently been identified in Xenopus laeuis, as well as a previously undescribed reorganization of envelope fibers occurring at fertilization. The molecular changes in the ECM glycoproteins (limited proteolysis, lectin-ligand binding, and conformational changes) and the oviductal and egg macromolecules responsible for the conversion of envelope forms are discussed. New experimental evidence that supports the lectin-ligand hypothesis for the formation of the fertilization layer is presented. It is proposed that the molecular changes in the ECM are responsible for the ultrastructural alterations of the ECM and for modifications of the fertilization and developmental functions of the anuran egg ECM. I t is not until the ouum has been clothed in the oviduct with its gelatinous envelope that it is susceptible of impregnation. G. Newport (1851)

. . .jelly-free body cavity eggs cannot be fertilized. , , . Whether the answer . . . rests within the jelly or with changes i n the egg, or with both, has not yet been determined. R. Rugh (1962)

INTRODUCTION The requirement of the ECM surrounding anuran eggs for fertilization was reported almost 140 years ago by George Newport (18511, who first observed sperm penetration of an amphibian egg. Newport’s observation that coelomic eggs are not fertilizable until they have passed through the oviduct has since been observed in many amphibian species. This observation remained unexplained for more than 100 years (Rugh, 1962). We now know that there are two reasons for the inability of meiotically mature coelomic eggs to be fertilized. First is the absence of jelly, which is added to the egg as it transits the oviduct. Factors in the jelly contribute to the sperm’s ability to penetrate the egg ECM. This was first shown by Kambara (1953) and has subsequently been demonstrated in many anurans (for references, see Metz, 1967). The second reason is the modification of the egg envelope that renders it penetrable to sperm. This was demonstrated in Rana pipiens by Elinson (1973), who chemically and physically

0 1991 WILEY-LISS, INC.

damaged the envelope t o render the egg fertilizable, and by Katagiri (1974) in Bufo japonicus. In vivo modification of the envelope ECM, which occurs in the oviduct, produces ultrastructural, chemical, and conformational changes in the envelope ECM (Grey et al., 1977; Nishihara et al., 1983). In addition, subsequent modifications of the envelope occur after the cortical reaction that render the envelope once again inpenetrable by sperm (Grey et al., 1976). Thus modifications of the anuran egg ECM are fundamental processes that

Received March 20, 1990; accepted in revised form July 24, 1990. Address reprint requests to Jerry L. Hedrick, Department of Biochemistry and Biophysics, University of California, Davis, CA 95616. Tatsuro Nishihara is now a t Suntory Limited, Mori Building 5-7-2 Kojimachi, Chiyodaku, Tokyo 102, Japan. Abbreviations used: CE, coelomic envelope; ECM, extracellular matrix; FE, fertilization envelope; F layer, fertilization layer; F material, solubilized F layer; HF layer, horizontal filament layer; OE, ovarian envelope; PR, pars recta; QDR, quick-freeze, deep-etch, replica; S layer, smooth layer; VE, vitelline envelope; VE*, VE-derived component of the FE.

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J.L. HEDRICK AND T. NISHIHARA

regulate sperm-egg interaction in fertilization. Understanding the structure and function relations of the anuran egg ECM is of biological significance, because fertilization, according to Lillie (19191, “is the central decisive event in the genesis of all sexually produced animals and plants.” The anuran egg is surrounded by two types of ECMs, the jelly coat layers and the egg envelope. The number of jelly coat layers, which are morphologically and chemically different, varies from one to five in different anuran species. We do not yet understand the significance of the variable numbers of jelly coat layers. The egg envelope is a single entity that exists in several related forms. In the case of Xenopus laevis, there are four distinct forms of the envelope: the ovarian envelope of the oocyte while it is still in the ovary, the coelomic envelope associated with the ovulated egg that is found in the coelom, the vitelline envelope of the oviposited egg, and the fertilization envelope associated with the fertilized egg or zygote. All four forms have unique ultrastructures; they also possess related yet different macromolecular compositions, except for the OE and CE, which have the same macromolecular composition. The chemistry of the anuran egg ECM differs from that of the somatic cell in that collagen and mucopolysaccharides are absent. The anuran egg ECM is composed of proteins and glycoproteins with no content of hydroxyproline or uronic acids. Additional organized structures in the ECM have recently been described in the perivitelline space of the X. laevis egg that are distinct from the envelope and jelly coat layers. These include the HF layer located on the microvillar tips in the perivitelline space of the oviposited egg and the derived S layer of the fertilized egg (for review, see Larabell and Chandler, this volume). The biological functions of the anuran egg ECM a t fertilization include 1) sperm binding, 2) induction of the sperm acrosome reaction, 3) a penetration barrier for the sperm (envelope penetration), 4) a block to polyspermy, and 5) provision of a protective environment for the developing embryo (normally referred to as envelope hardening). These functions are, in general, the same for the ECMs of all animal egg. There is a n additional developmental function of the ECM. At some stage of development, the embryo must hatch from the ECM. The hatching process is chemomechanical in nature and requires the release of a hatching enzyme that partially hydrolyzes the ECM. This enzymatic hydrolysis is coupled with vigorous thrusting movements that permit the embryo to escape from the protective ECM and to continue development as a freeliving tadpole. Recent investigations into the ultra- and chemical structures of the anuran egg ECM have provided additional understanding into their function. In this paper we will review recent findings, primarily from our own laboratory using X . laeuis, and provide some new observations on the structure-function relations of the anuran egg ECM. Other relevant reviews include those by Katagiri (1987), Dumont and Brummett (1985), and Schmell et al. (1983); relevant studies in urodeles have been reviewed by Elinson (1986).

MATERIALS AND METHODS Hormonal Induction of Ovulation X . laevis was maintained on a 12 h light-12 h dark cycle. Hormone stimulation of anurans for the production of gametes has been used since the original observations of Houssay et al. (1929) and Wolf (1929), who used pituitary explants. In general, anurans release gametes in response to injection of pituitary extracts from other amphibians but not to mammalian hormones. Only the pipid X . laevis and Bufo species respond to mammalian pituitaries or gonadotropins (for discussion, see Rugh, 1962; Deuchar, 1975). The LH activity of human chorionic gonadotropin has traditionally been used to induce ovulation, but we find that the FSH activity of pregnant mare serum gonadotropin is also beneficial. FSH- and human chorionic gonadotropin-injected females generally produce more eggs of a higher quality, sooner after injection, and on a more predictable schedule than those injected with human chorionic gonadotropin alone. Impure preparations of the glycoprotein hormones can be used. Pregnant mare serum gonadotropin stimulation of females is also beneficial, as i t increases the amount of oviductal secretions that can be extracted from the oviduct (Hardy and Hedrick, unpublished observations). Pregnant mare serum gonadotropin was previously used in laeuis to increase the number of vitellinogenic oocytes for studies on vitellogenin synthesis (Redshaw and Nicholls, 1971; Follett and Redshaw, 1974). Conditions found to be optimal for egg production are the following (see also Fig. 1). Females were injected into the dorsal lymph sac with 35 IU of pregnant mare serum gonadotropin dissolved in isotonic saline. After 4 days, they were injected with 500-1,000 IU of human chorionic gonadotropin, and, 5-6 h post human chorionic gonadotropin, eggs were stripped from the females three to four times a t 1.5-2 h intervals into DeBoers solution (110 mM NaC1, 1.3 mM CaCl,, 1.3 mM KC1, pH 7.2, with NaHCO,). The number of eggs collected was determined volumetrically (150-200 jellied eggsiml).

x.

Isolation of Egg Envelopes Egg envelopes were prepared from coelomic of dejellied oviposited eggs by sieving with nylon screens as previously described (CE, Gerton and Hedrick, 1986a; FE, Gerton and Hedrick, 198613; VE, Wolf et al., 1976). The OE was isolated from oocytes released from ovaries with a meat grinder. The oocytes were separated from tissue debris using large-mesh nylon screens. The OE was isolated from homogenized oocytes by sieving with nylon screens (Hardy and Hedrick, unpublished observations). Solubilization of the F layer from isolated FEs utilized either Gal- or EDTA-containing solutions to reverse the cortical granule lectin-ligand reaction, which is responsible for the formation of the F layer (Wyrick et al., 1974). A suspension of 10,000 isolated FEs was extracted in 3 ml of 500 mM Gal, 10 mM Tris-HCL, 154 mM NaC1, and 1 mM CaCI,, pH 7.8, for 2 h a t 22”C, followed by washing (centrifugation at 5,OOOg for 5 min) four times with 3 ml of ice cold water. Alterna-

ANURAN EGG EXTRACELLULAR MATRIX I

'

I

'

I

'

I

'

I

321

Pan Convduta

-

Coelom

ovary

Fig. 2. The tissue and cellular sites of the synthesis or modification of the anuran egg ECM. The route of the egg from ovarian release to oviposition and subsequent fertilization is illustrated.

0

*

2

lo l2 l4

l 6 l8

.

Days P o s t PMSG I

0

20

I

I

40

I

I

I

60

I

80

I

I

100

I.U. PMSG Fig. 1. Egg production response as a function of pregnant mare serum gonadotropin (PMSG) dose. Varying amounts of PMSG were followed 4 days later by a n ovulating dose of 1,000 IU of human chorionic gonadotropin. Inset: Egg production as a function of PMSG postinjection time. Eggs were collected and counted as described in Materials and Methods. Maximum response was 2,400-3,300 eggs per frog. Each data point was the average of the eggs obtained from 5-10 frogs.

tively, the isolated FEs were extracted with 5 mM EDTA, 10 mM Tris-HCL, 154 mM NaCI, pH 7.8. The soluble FE extract (F material) was combined with the envelope washings and extensively dialyzed against H,O, as were the particulate VE*, to remove Gal (EDTA). The dialyzed F material was concentrated by rotary evaporation (35°C) to 1.2-2.0 ml. The concentrated F material was again dialyzed against 1 mM EDTA, 10 mM Tris-HC1, pH 7.5, to remove any residual calcium ions. Typically, 1 mg of F material protein was recovered from 10,000 FEs. The solubilized F material was quantitatively precipitated by the addition of 1.34 mM CaC1, or DeBoers solution. For the reconstitution experiments, 300 pg (protein) of F material in 0.75 ml of 1 pM EDTA was added to 1,000 isolated, washed VE* (or VE) and the suspension incubated for 5 min a t 22°C. Then 0.1 volume of 10 x DeBoers solutions was added and the suspension quickly mixed. The suspension was incubated for 30 min at 22°C; the reconstituted envelopes were removed by filtration on a 102 pm nylon screen, washed with a n ice-cold 1.34 mM CaC1, solution, and fixed for electron microscopy.

Electron Microscopy Eggs were fixed for 1 h at room temperature in 2.5% glutaraldehyde, 0.1% tannic acid (added immediately before use), 2% sucrose, 0.05 M sodium cacodylate, pH 7.4. The fixed eggs were rinsed and postfixed for 1.5 h in 2% OsO,, pH 8.0. After rinsing, they were dehydrated using acetone and embedded in epoxy resin (Ted

Pella, Inc.). Thin sections were stained with lead citrate and uranyl acetate. Isolated envelope components, e.g., VE*, were fixed for 2 h in ice-cold 3% glutaraldehyde, 0.1 M sodium phosphate buffer, pH 7.2. Envelopes and envelope components were recovered by centrifugation, rinsed with phosphate buffer, and then postfixed for 1 h in 1%OsO, in 0.1 M sodium phosphate buffer, pH 7.2. The fixed material was collected by centrifugation. dehvdrated in ethanol. and embedded in Spurr's plastic. Thin sections were stained with lead citrate and uranyl acetate.

RESULTS AND DISCUSSION Sites of ECM Synthesis and Modification Formation of the envelope takes place during oogenesis. Using antibodies against the VE and immunocytochemical methods, the biosynthesis of the OE glycoproteins was found to occur solely in the oocyte itself and was first detected in previtellogenic late stage I oocytes in X . laeuis (Yamaguchi et al., 1989). The deposition of the fibrous OE in the interstices of the follicle and oocyte microvilli is not discernible until early stage I1 (Dumont, 1972). The biosynthesis and secretion of the OE glycoproteins appears to involve higher molecular weight precursor molecules (more highly glycosylated precursors?) and does not involve membrane-bound transport vesicles or granules. The subcellular site of OE glycoprotein synthesis is likely to be the Golgi apparatus, but this has not yet been experimentally shown. Figure 2 illustrates the path of ovulated eggs and the tissue or cellular sites where the ECM is modified. The mature oocyte is ovulated into the coelom or body cavity and is swept by ciliary action into the ostium of the oviduct, where it enters the pars recta region of the oviduct. The PR oviduct has a gross anatomy that is different from the pars convoluta oviduct (Rugh, 1935) and has distinct histological and cytological structures (for Rana japonica and X . laeuis, see Katagiri et al., 1982; Yoshizaki, 1985; Yoshizaki and Katagiri, 1981). In X. laeuis, Yoshizaki (1985) has separated the approximately 2 cm PR oviduct into two regions based on the cytology of the secretory cells. The ultrastructural rearrangement of the envelope fibers that is a part of the CE to VE conversion was originally observed in our laboratory in the PR oviduct using X . laeuis (Grey et al., 1977). This conversion takes place in the first 1 cm of the PR oviduct (PR1 according to Yoshizaki's nomen-

322


clature). The CE to VE conversion has also been observed in R. japonica (Yoshizaki and Katagiri, 19811, B.japonicus (Katagiri et al., 1982), and Bufo arenarum (Mariano et al., 1984). Yoshizaki (1985) states that in X . laeuis, 8 nm particles are also added to the envelope as it passes through the PR oviduct, although Grey et al. (1977) interpreted this observation as due to cross sections of envelope fibers. Such particles were not observed using QDR methods (Larabell and Chandler, 1988). Resolution of the differences in interpretation of the VE transmission electron microscopic observations clearly requires some additional experimental evidence. In X . laeuis, the lightly staining cloud-like prefertilization layer, originally described by Yoshizaki and Katagiri (19841, is added to the outer aspect of the envelope in the PR2 oviduct (Yoshizaki, 1984). The recently described large meshed and netlike HF layer is added to the perivitelline space and is likely a secretion product of the PR oviduct, because i t is not present in the coelomic egg, but is present in the oviposited egg (Larabell and Chandler, 1988, 1989b; see also Larabell and Chandler, this volume). Thus secretions of the PR oviduct are involved in two and possible three ultrastructural alterations of the ECM: 1) modification of the fibrous structure of the envelope, 2) creation of the PF layer and, possibly, 3 ) construction of the HF layer. The X . Zaeuis pars convoluta oviduct secretes three or four jelly coat layers that are sequentially wrapped around individual eggs as the eggs are transported through the oviduct (del Pino, 1973; Freeman, 1968; Yoshizaki, 1985). Yoshizaki (1985) identified four histologically distinct regions of the approximately 20 cm long pars convoluta and a fifth region that constituted the ovisac (uterus) where the fully jellied eggs are stored prior to oviposition. In some anurans (more often toads than frogs) jelly coat layers can be continuously applied to a clutch or batch of eggs rather than individually wrapped around each egg (Salthe, 1963). For instance, in B . japonicus two jelly coat layers are individually wrapped around each egg and two more jelly coats are wrapped around the egg clutch in a continuous fashion, giving rise to a string of jellied eggs (Katagiri, 1965; Kobayashi, 1954). After oviposition, the eggs must be fertilized within a short period of time before the jelly coats imbibe water and swell or before water-soluble components necessary for fertilization diffuse from the jelly (for discussion, see Katagiri, 1987). Water imbibition by the jelly impairs fertilization and presumably causes a n as yet undefined structural reorganization of the jelly coat layers. Sperm penetrating through the jelly coat layers undergo the acrosome reaction in the innermost jelly coat layer (light microscopic observations) near or a t the outer surface of the VE in the two anurans that have been studied (Leptodactylus chinquensis, Raisman and Cabada, 1977; B . japonicus, Yoshizaki and Katagiri, 1982). Ultrastructural studies on in situ anuran sperm acrosome reactions, penetration of the egg envelope, and anuran sperm-egg membrane fusion events have yet to be recorded. The sperm-induced cortical reaction converts the VE into the FE, first described in X . Zaeuis (Grey et al., 1974), along with other

pronounced changes in the cell surface (e.g., Balinsky, 1966). The ultrastructual modifications of the envelope include the formation of a n F layer on the outer surface of the VE from its precursor PF layer and reorganization of the envelope fibers into concentric fibrous sheets. In addition, formation of the S layer derived from the H F layer occurs in the perivitelline space (Larabell and Chandler, 1988). All of these ultrastructural changes involve reactions of the egg ECM with macromolecules released from the cortical granules in the sperm-induced cortical reaction.

Ultrastructure and Macromolecular Composition of Jelly Coat Layers The fine structure of the jelly coat ECM is inadequately described. The jelly coat layers of a number of anuran eggs have been examined by light microscopy, but this has revealed only the number of jelly coat layers present, although not always in a convincing manner, and provided some information on the cytochemical composition of the jelly (for review, see Dumont and Brummett, 1985). One electron microscopic study on the jelly coat layers of X . laeuis has been reported but at very low resolution so that the fine structure of the jelly coats are not apparent (Yoshizaki, 1985). Electron microscopic studies of the jelly coats using methods appropriate to the hydrated nature of the ECM such a s freeze substitution and QDR methods are needed to reveal the fine structure of this biologically important ECM. The macromolecular composition of the individual jelly coat layers has been determined only for X . laeuis (Yurewicz et al., 1975). The three jelly coat layers (41 pglegg) were individually isolated and their macromolecular compositions determined by five different analytical methods. The three layers are composed of eight to nine highly glycosylated glycoproteins in total. Each layer apparently has a unique composition, and the glycoproteins composing a n individual layer are not uniformly distributed within that layer. The innermost layer, J1,contains sulfate. Sulfate, in ester linkage, is a common component for amphibian jelly in general and is usually associated with the innermost jelly coat layer (for references, see Hedrick et al., 1974). In the case of X . laeuis, it was suggested on the basis of sulfate ester hydrolysis kinetics and infrared spectrophotometry observations that sulfate esters of secondary axial hydroxyl groups of Gal, GalNAc, andlor Fuc were present (Hedrick et al., 1974). The common chemistry and structural location of sulfate in the innermost jelly coat layer implies a functional importance to glycoprotein sulfate esters. Isolation and characterization of molecules composing the jelly coat layers, sulfated and nonsulfated alike, have provided a substantial challenge to the biochemist because of the poor solubility characteristics, extreme aggregation properties, highly glycosylated nature, and fibrous shape of jelly coat macromolecules. The chemical and structural properties of the jelly macromolecules need to be thoroughly investigated because of their biological importance in anuran fertilization: “In view of the essential role of amphibian jelly in fertilization, the chemistry of this material should be of unusual interest” (Metz, 1967).

ANURAN EGG EXTRACELLULAR MATRIX

The essential role of the jelly coat layers in anuran fertilization is well documented, but mechanistically ill defined (for discussion, see Katagiri, 1987). In Discoglossus pictus, a specialized portion of the jelly, a lens-shaped plug, seems to function by focusing the penetration path of sperm toward the animal dimple, where envelope penetration and sperm-egg plasma membrane fusion takes place (Talevi and Campanella, 1988). The most definitive study on the function of the jelly coat is that of Ishihara et al. (1984). Using B. japonicus, they concluded that the function of the egg jelly was to bind Ca2+ andlor Mg2+,thereby providing environmental conditions necessary for the sperm acrosome reaction and for successful sperm penetration of the envelope. Dejellied eggs could be fertilized using a defined solution of salts without addition of jelly or jelly-derived substances (Katagiri, 1986, 1987). However, even though this is the first experimentally supported explanation for the functional requirement for jelly in anuran fertilization, our understanding of the process is still incomplete, a s the molecules involved in the binding of Ca2+/Mg2+have not yet been identified or the chemical nature of the binding process defined. The substitution of a balanced salt solution for the jelly coats and the successful fertilization of dejellied eggs is limited to B. japonicus It has not yet been reported for any other anurans. Accordingly, we do not yet know the general applicability of Ca2+ andlor Mg2+ binding properties of jelly coat macromolecules or if this metal-binding property is the one jelly coat function essential for anuran fertilization. From a teleological point of view, it is difficult to explain the multiplicity of the jelly coat layers, their species-specific properties, and the complexity of their macromolecular composition, if metal binding is the only function that is essential to anuran fertilization. It has recently been observed that some of the jelly coat macromolecules of X . laevis and B. japonicus eggs are immunologically related. One of these common antigens can function as a ligand for the X . laevis cortical granule lectin, which is released in the sperm-triggered cortical reaction (Hedrick and Katagiri, 1988). Considering that these two organisms are systematically far removed from one another, having been classified as 70 million years apart in geological time (Duellman and Treub, 1986), some of the jelly coat macromolecules in these two species are evolutionarily highly conserved and therefore should play some essential roles in anuran fertilization. A comparative immunological approach was previously used with anuran jelly, but it did not lead to significant understanding of the jelly’s role in fertilization because this approach was not coupled with other experimental approaches (for discussion, see Metz, 1967). A comparative approach to the structure-function properties of the egg jelly coat layers that couples molecular, immunological, and ultrastructural studies may provide penetrating insights into the function of jelly ECM in the fertilization process. The value of a comparative approach to understanding biological processes was ably presented by Tyler (1967), particularly a s applied to gamete biology.

323

Ultrastructure and Macromolecular Composition of Egg Envelopes Ovarian Envelope. As stated earlier, the macromolecular biosynthesis of the envelope begins in late stage I oocytes, and molecular assembly into a visible extracellular structure occurs in stage I1 oocytes. Formation of the envelope is largely completed by stage V of 00genesis (Dumont, 1972). The meiotically mature stage VI oocyte is surrounded by a n envelope that has a n ultrastructure distinct from that of the ovulated coelomic egg (Dumont and Brummett, 1977; Grey et al., 1977). As determined by transmission electron microscopy, the dominant substructure of the envelope is that of loosely packed fibers, 4-7 nm in diameter, bundled together, giving the envelope the appearance of coarse filter paper (Fig. 3). Embedded within the OE are microvillar processes emanating from the egg surface and macrovillar processes extending from the follicular cells. This columnar or radial appearance of the OE in cross section gave rise to one of its former names, the zona radiata (Wischnitzer, 1966). The surface of a n isolated stage VI oocyte as seen by scanning electron microscopy has a doormat-like appearance of fibrous bundles with pores leading into tunnels where the macrovillar processes formerly permeated the envelope. The tunnels are likely produced when the envelope matrix is formed by the assembly of fibers around villar processes (Dumont and Brummett, 1977). Both the follicle cell and oocyte villar processes a r e retracted from the envelope before ovulation. More recent studies by Larabell and Chandler (198913) using QDR methods provide a n expanded and refined description of the OE, with greater definition of the fiber content of the envelope, although the general structural characteristics of the envelope are as described above. The OE is composed of three different fibers that differ in size and topological position. The largest fibers are approximately 15 nm in diameter (which includes the platinum thickness of approximately 1.6 nm) and are grouped together into fibrous bundles that have diameters of 75-100 nm. Thus the bundles are formed from some 25-44 individual fibers. These bundles undoubtedly correspond to those previously observed in the OE and CE and described as 40-70 nm in diameter. The fibrous bundles are less tightly packed, and the fiber density is more uniform in the outer margin (follicle cell surface) than in the inner margin (oocyte surface) of the envelope. The fibrous bundles are connected to the envelope permeating egg microvilli with intermediate sized fibers of 8 nm diameter. These intermediate fibers are in turn interconnected with small fibers of 4 nm diameter. Larabell and Chandler (1989b) have provided the first description of the ECM of the perivitelline space. Previous transmission electron microscopic studies indicated the presence of structures within the perivitelline space, but the methods used did not permit a coherent and organized perception of this extracellular space (an unfortunate historical choice of words, as the word implies a place devoid of structure). The perivitelline space of the mature oocyte consists of a dense network of fibers that connect the microvilli with the

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Fig. 3. TEM micrograph of the ovarian oocyte envelope. CG, cortical granule; PS, perivitelline space. Bar = 0.2 pm; x 38,000. Fig. 4. TEM micrograph of the coelomic egg envelope. CG, cortical granule; PS, perivitelline space. Bar = 0.2 pm; X 38,000.

Fig. 5. TEM micrograph of the oviposited egg envelope. Bar = 0.2 pm: x 34,000. CG, cortical granule; PL, prefertilization layer; PS, perivitelline space. Fig. 6. TEM micrograph of the fertilized egg envelope. Bar = 0.2 pm: x 26,000. F,, condensed portion of the fertilization layer: F,j,dispersed portion of the fertilization layer; PS, perivitelline space.

325

ANURAN EGG EXTRACELLULAR MATRIX TABLE 1. The macromolecular composition of isolated X. laevis envelopes OE gp120

CE

VE

FE hi-MW gp120 (6.5

gp120 (6.2

f

1.5)

gp112 gP69

gp120 (6.2) gp112 (6.4) gp69 (1.4)

gp112 (6.5 gp69 (1.9

f -t

1.4) 0.8)

gP64

gp64 (1.9)

gp64 (2.6

F

0.6)

gp57 (0.9

f

1.2)

gP43

$343

a37

gp37 (37)

gp112 (8.5

Polypeptide MW (K)

N.D. f 0.9) F 1.8)

gp66 (2.2 f 0.4) gp61 (3.2 ? 1.4) gp57 (0.1 2 0 . 1 ) gp45-40

(47) gp41 gp37

( 4 3 k 2) (39 k 5)

gp41 (43 i 4) gp37 (37 i 1)

83,78 &75 54 52 54 52 57 39 38 36 36

Components resolved by 6-8.5% SDS-PAGE (protein stained) after disulfide bond reduction of the samples. Previous nomenclature denoted the envelope components by their apparent molecular weights (see, for instance, Gerton and Hedrick, 1986a).The numbers in parentheses represent the percentage -t SD of the total envelope components obtained from image analysis of Coomassie blue-stained gels. The FE analysis for percent composition was based on the VE*. The MW of the polypeptide moiety of the glycoproteins was determined after chemical deglycosylation (Lindsay and Hedrick, 1989; Wardrip and Hedrick, 1989; Bakos et al., 1990a).

oocyte surface, interconnect the microvilli, and inter- been studied in as much detail (R.pipiens and Rana digitate with the fibers of the envelope (for discussion, clamitans, as cited in Grey et al., 1977; R. japonica, Yoshizaki and Katagiri, 1981; B.japonicus, Katagiri et see Larabell and Chandler, this volume). For molecular studies, the OE can be isolated en al., 1982; B. arenarum, Mariano et al., 1984). In the X . masse by disrupting the ovary with a meat grinder and laevis CE, the fibers are packed into bundles more separating the oocytes from other cells and cellular de- loosely than in the OE, according to Larabell and bris by filtration. The washed oocytes are ruptured and Chandler (1989a). The CE has a more spacious or open the OEs isolated using filtration through nylon screens structure than the OE. The dimensions of the individa s with the VEs and FEs (Gerton and Hedrick, 1986a,b; ual fibers in the CE, however, remained unchanged Wolf et al., 1976). The macromolecular composition of from those of the OE. The outer surface looks like a n the OE is the same as the CE, a s determined by SDS- aerial view of California freeway interchanges as the PAGE, i.e., six glycoproteins can be detected (Table fibrous bundles crisscross one another and merge. Du1).Oocytes were also released from the ovary using mont and Brummett (1977) suggested, based on scancrude collagenase preparations and the OE isolated ning electron microscopic observations, that the outer from these oocytes. However, the gp43 component of surface of the CE appeared smoother than that of the the OE was partially hydrolyzed by the protease con- OE. However, the scanning electron micrographs of taminants in the crude collagenase and converted to a Grey et al. (1977) and the QDR micrographs of Larabell glycoprotein that appeared to be identical to the gp41 and Chandler (1989a) show a very coarse net- or matcomponent, a molecule characteristic of the VE. Inhi- like surface to the CE that is not different from the OE. bition of the protease contaminants in the crude colla- Perhaps the observations of Dumont and Brummett genase, using diisopropyl fluorophosphate to prevent were due to the “tearing away” of the follicle cells from hydrolysis of the gp43 component, was not effective, as the ovarian envelope surface with forceps in contrast to such inhibited collagenase preparations did not disso- the physiological withdrawal of the macrovillar prociate the ovary into its constituent cells. It was surpris- cesses and separation of the follicular cells from the ing that other OE components were not susceptible to envelope prior to ovulation. Changes in the perivitelline space ECM also apparhydrolysis by the protease contaminants-only the gp43 component. We have subsequently shown, using a ently reflect the reorganization of the oocyte surface. variety of endoproteases, that the gp43 component is The retraction of the microvilli and macrovilli from the apparently “exposed” to the bulk solution in a way that envelope produces a n array of oblique fibers in the is different from other OE glycoproteins (Hardy and perivitelline space that interconnects microvilli with the cell surface, with each other, and with the envelope Hedrick, unpublished observations). Coelomic Envelope. The ovulated coelomic egg is (see Larabell and Chandler, this volume). The CE has been isolated from X . laevis coelomic devoid of follicle cells and the microvillar processes rising from the egg surface are substantially shorter. The eggs and its physiocochemical properties studied ultrastructural changes that occur in the envelope (Gerton and Hedrick, 1986a; Grey et al., 1977). To obupon ovulation appear to be a response to the with- tain coelomic eggs, the oviduct was ligated via a small drawal of the villar processes by the oocyte and follicle incision in the body wall prior to hormone stimulation. cells. The large fibrous bundles or filamentous fascicles After ovulation, coelomic eggs could be readily recovof the CE, first described for X . laevis in our laboratory ered from the body cavity. The CEs were obtained from (Grey et al., 1977) and seen in the ovarian oocyte OE by the lysed eggs by filtration through nylon screens (Wolf Dumont (1972), remain the dominant feature of the et al., 1976; Wyrick et al., 1974). The macromolecular envelope (Fig. 4). The ultrastructure of the CE has composition of the CE was the same as that of the OE, been observed in several anuran species, and the gen- being composed of six glycoproteins with apparent moeral structural features of the envelope appear to be lecular weights between 37K and 120K (Table 1).An similar to those in X . laevis, although they have not SDS-PAGE protein-stained gel of the B. japonicus CE

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appears very similar to that of X . laevis CE (Takamune et al., 1986). Unfortunately, definitive results on the macromolecular composition of the CE obtained from the eggs of other anuran species are not available. Such information would greatly assist the attempts to understand the biological properties of the egg envelope, since isolated CEs retain their ultrastructural and biological properties and cannot be penetrated by sperm (Grey et al., 1977),just as eggs recovered from the coelom cannot be fertilized even when supplemented with egg jelly: Vztellzne Envelope. After the egg passes through the PR oviduct, the envelope is converted to a form characteristic of the oviposited egg (Grey et al., 1977). A marked ultrastructural change in the egg envelope of X . laevis accompanies the change from sperm impenetrable to sperm penetrable. The individual fibers of the fibrous bundles are dispersed rather evenly within the boundaries of the envelope and arranged parallel to the egg surface (Fig. 5). The tunnels that were present in the CE are now lost, and the envelope has a much more uniform appearance in terms of fiber distribution. The external surface is also more uniform and is relatively smooth compared with the CE; the thickness of the envelope is unchanged from that of the CE. Recent measurements of fiber diameters suggest that the diameter of the largest fibers, which were organized into bundles in the CE, increases (15 ? 1.6 to 19 1.6 nm; the platinum coating is included in these measurements), while the other smaller and less numerous interconnecting fibers with diameters of 8 and 4 nm remain unchanged (Larabell and Chandler, 1989a). Thus the major structural element of the envelope, the bundled 15 nm fibers, is altered when the CE is converted to the VE in the PR oviduct. Similar ultrastructural changes occur in other anuran envelopes based on transmission electron microscopic studies (R.japonica, Yoshizaki and Katagiri, 1981; B. japonicus, Katagiri et al., 1982; B. arenarum, Mariano e t al., 1984), but more definitive studies using QDR methods need to be done in these anurans. A new architectural feature is evident in the perivitelline space in X . laevis eggs after passing through the oviduct (Larabell and Chandler, 198913). A layer of interconnecting fibers is added onto the tips of the microvilli that extend into the perivitelline space. This HF layer is composed of 5 and 10 nm diameter fibers and has the appearance of a fine-meshed net. The H F layer is in intimate contact with the innermost aspect of the VE. As mentioned earlier, it seems likely that the H F layer is derived from substances secreted from the PR oviduct that are assembled to form the H F layer as the egg transits the PR oviduct. The function of the HF layer is unknown, but its structure is modified a t fertilization (discussed in the next section). The other fibers in the perivitelline space, the oblique fibers, that interconnect microvilli with the cell surface, with each other, and with the envelope, are apparently unchanged. Analogous observations in other anuran eggs have not been reported. The VE from dejellied oviposited X . laevis eggs has been isolated and its physicochemical properties studied (Gerton and Hedrick, 1986a,b; Nishihara et al.,

*

TABLE 2. Physical properties Tm("C) Solubilization by trypsin Solubilization by mercaptoethanol Accessible sulfhydryl groups (nmolimg protein) Accessible Tyr resides (iodination of gp43igp41) ANS binding Affinity (Kd x M) No. sites (mmolimol protein) Ferritin binding (kgimg protein) Symmetrical F layer binding Deformability (krnil0' dynesicm?

of

isolated X . laevis envelopes

CE

VE

FE

References

51 ND ND

42

+ +

56 -

1 2 3

92

140

130

1

+

-

-

4

7 430 275 ND 13

47 510 59

74 650 41

1 1 1

25

1

5 1

+

-

ANS. 1-anilino-8-nauhthalene sulfonic acid. ND.. not determined: Tm. melting ~

temperature. References are 1, Bakos et al. (1990a); 2, Wolf (1974); 3, Wolf et al. (1976); 4, Nishihara et al. (1983) and Bakos et al. (1990b); 5, this paper.

1983; Wolf et al., 1976). It is unknown if the intimately associated H F layer remains with the egg surface or is attached to the inner surface of the isolated VE. The isolated VE is composed of seven glycoproteins as noted in Table 1 (the gp57 component was previously classified as a protein; recent observations suggest it is a glycoprotein; H. Fabry and J.L. Hedrick, unpublished). One of the CE glycoproteins, gp43, is converted via limited proteolysis into gp41. The component added to the envelope, gp57, is presumably a secretion product of the PR oviduct, although this has not been established yet; perhaps the gp57 component is the molecule assembled to form the H F layer. The physical properties of the VE are markedly different than those of the CE from which it is derived (Table 2). The altered physical properties of the envelope likely result from a rearrangement of the glycoproteins composing the envelope. This rearrangement probably involves conformational changes of some glycoproteins (gp43?) that change their interactions with other glycoproteins. The integrated conformationallrearrangement changes likely produce the altered ultrastructural and the altered biological properties of the egg envelope. However, a logically satisfying hypothesis that integrates the molecular, ultrastructural, and cell biological properties of the X . laevis egg envelope is riot possible yet. The macromolecular composition of the VE from other anuran eggs has not yet been determined. As with the CE, a n SDS-PAGE gel of the VE from B. japonicus eggs appears very similar to that of the X . laevis VE (Takamune et al., 1986, 1987). A group of glycoprotein(s) with a n MW range of 39K to 52K are converted to glycoproteins with MWs of 36K and 39K in B . japonicus, analogous to the gp43 to gp41 conversion in X . laevis. As will be discussed later in this section, the glycoproteins that are altered in the CE to VE conversion in B. japonicus and X . laevis are immunologically related (Takamune et al., 1987). The limited hydrolysis of glycoproteins in B. japonicus and X . laevis CE to VE conversions noted above suggests that a hydrolase is released from the PR oviduct that specifically hydrolyzes one or two envelope glycoproteins. The first observations of a protease extracted from the PR oviduct that altered the fertiliz-

327


ability ofB. arenarum coelomic eggs was that of Miceli e t al. (1978a,b). Cabada et al. (1978) also showed that trypsin inhibitors and concanavalin A inhibited the action of the PR extract on the coelomic eggs. It was subsequently shown that a partially purified enzyme from the PR oviduct was a glycoprotein (concanavalin A reactive), had “trypsin-like” specificity toward synthetic substrates, rendered the CE more susceptible to hydrolysis by B. arenarum sperm lysin, altered the envelope’s hydrophobic dye-binding properties, and changed the ultrastructure of the CE to that of the VE (for review, see Miceli, 1986). Attempts to identify the envelope glycoprotein hydrolyzed by the enzyme were unsuccessful as the purified PR fraction dissolved the CE. This observation is troublesome in view of the similar enzymatic properties of the B. japonicus and X . laevis enzymes (discussed below) and because the egg envelope is likely in continual contact with the protease after it has been secreted from the oviduct and added to the envelope. Obviously, for the envelope to fulfill its biological functions in fertilization and development, it must remain particulate or insoluble, and frank solubilization by a PR protease seems detrimental. Perhaps the envelope-dissolving property of the isolated B. arenarum PR fraction was due to contamination by other proteases. In B. japonicus, Takamune et al. (1986) and Takamune and Katagiri (1987) highly purified a protease from the secretory granules of the PR oviduct and determined the properties of the enzyme. The protease was specific for Arg residues (using synthetic substrates), had a pH optimum of 8.0-8.2, was inhibited by serine protease inhibitors, irreversibly inhibited by EDTA, and had a n MW of 66K. The protease specifically hydrolyzed the 39K to 52K CE glycoproteins with the concomitant release of small molecular material (peptides) and rendered the envelope susceptible to sperm lysin solubilization. The enzyme did not hydrolyze other envelope components, and it did not solubilize the envelope. The impure enzyme (PR granule extract) rendered coelomic eggs fertilizable (reaction inhibited by concanavalin A), but the purified enzyme was ineffective, even though limited proteolysis of the 39K to 52K glycoproteins occurred. Takamune et al. concluded that the PR protease was required, but not sufficient for rendering coelomic eggs fertilizable. Katagiri and colleagues previously demonstrated the presence of acrosome-inducing substances in the PR granule extract (Katagiri e t al., 1982; Yoshizaki and Katagiri, 1982). Takamune et al. (1986) noticed the appearance of a 170K molecule in their PR extracttreated CEs that was also concanavalin A reactive. They suggest that the 170K molecule might be the sperm acrosome reaction-inducing factor that they postulate must be present with the PR protease in order to fertilize coelomic eggs. Characterization of a PR protease from the oviducts of X . laeuis have utilized both PR fluid obtained from ligated oviducts (Bakos et al., 1990a,b) and extraction of purified PR granules (Takamune et al., 1987; Takamune and Katagiri, 1987). The PR proteolytic activity had properties similar to those of B. arenarum and B. japonicus PR enzymes, being specific for Arg residues

(synthetic substrates) and inhibited by serine active site reagents. Treatment of the X . laevis PR fluid with [32Pl-diisopropyl fluorophosphate yielded three radioactive labeled molecules, one of which had a n MW of 68K. The PR fluid specifically catalyzed the conversion of the gp43 of the CE to gp41 by proteolysis a t the C-terminal end of the polypeptide chain and did not hydrolyze other envelope components, and it did not solubilize the envelope. The enzyme also altered the melting temperature of the CE, the accessibility of the CE gp43 component for iodination, the envelope’s ferritin-binding properties, and the fertilizability of coelomic eggs. The PR protease has recently been purified 33-fold from a 12,OOOgpellet of a PR extract by affinity chromatography on p-aminobenzamidine-Sepharose and by HPLC using hydroxylapatite (Hardy and Hedrick, 1989; unpublished observations) and characterized as a n enzyme and as a protein. The purified protease, designated as oviductin in view of its source, is specific for Arg peptide bonds (synthetic substrates), has a pH optima of 8, is stimulated about twofold by 0.2 M NaC1, is inhibited by serine active site reagents, and is stabilized against autolysis by Ca2 . It specifically hydrolyzed the CE gp43 to gp41 and altered the melting temperature of the CE to that of the VE. The Nterminal amino acid sequence (28 residues with six introduced gaps to maximize amino acid identity) was homologous with several other serine active site proteases, e.g., 64% identity with spiny dogfish trypsin. The invariable N-terminal residues of serine active site proteases (Gly4, Prol3, Cys32, Gly33) were all present in the oviductin sequence. The amino acid residue a t position 35 is often a Ser or Thr, and we obtained a “ b l a n k residue a t this position during the sequence determination. One possible explanation for such a blank amino acid residue is a glycosylated SerlThr; this seems possible in view of the concanavalin A reactivity of B . arenarum and B . japonicus proteolytic activities and by analogy with sperm acrosin, which is a glycosylated serine active site protease (for discussion, see Hedrick et al., 1989). Purified oviductin will convert the ultrastructure of the CE so that it is similar to that of the VE as determined by transmission electron microscopic methods (Larabell et al., 1989a). From the observations of Larabell and Chandler (1989b), the bundled 15 nm fibers of the CE are dispersed into separate 19 nm fibers in the VE. Perhaps this fiber conversion and dispersion reflects the gp43 to gp41 conversion catalyzed by oviductin if the 15 nm fibers are -partially or totally composed of gp43. The similarity of the properties of oviductin from X . laevis, B. japonicus, and B. arenarum suggests that the proteolytic process for converting the CE to the VE might be a n evolutionarily highly conserved mechanism. Experiments with X . laevis and B. japonicus support this suggestion (Takamune et al., 1987). As mentioned earlier, the CEs from the two species contain glycoproteins that are immunologically related; these are the glycoproteins that undergo limited proteolysis in the CE to VE conversion. The oviductin from one species not only catalyzed the limited proteolysis of its own envelope glycoprotein, it also catalyzed the limited proteolysis of the other species envelope glycoprotein, +

328


i.e., the proteases hydrolyzed both the species homologous and heterologous substrates. Thus highly specific and limited proteolysis of a n envelope glycoprotein is apparently a fundamental mechanism underlying the alteration of the ultrastructure and function of the envelope ECM. Although this alteration is essential to the fertilization process, other macromolecules secreted by the PR oviduct (as well as by the pars convoluta oviduct) are required for successful sperm-ECM interaction in anurans. As stated earlier, two and possibly three ultrastructural modifications occur to the ECM as the egg transits the PR oviduct. One of the functions of the VE, a s mentioned in the Introduction, is to provide binding ligands for sperm receptors. Attempts to identify a VE component involved in sperm binding with X. laevis utilized radiolabeled, solubilized VE components (Gerton et al., 1982) and sperm extracts (SDS). Interaction of the sperm-VE components was detected using blotting methods (Lindsay and Hedrick, 1988). Blotted sperm components with apparent MWs of 35K, 25K, 19K, and 14K bound the solubilized [12511-heat solubilized VE components. The sperm components were found to be localized on the sperm surface as shown by solid phase radiolabeling of the sperm surface. Of the SDS-PAGE separated, blotted VE components, only the gp37 component interacted with solubilized radiolabeled sperm components. Chemically deglycosylated gp37 also interacted with sperm components, suggesting that the carbohydrate moiety of the glycoprotein was not important. However, selected sulfated carbohydrates, e.g., fucoidin and dextran sulfate, inhibited fertilization and the binding of heat solubilized VE to blotted sperm components, suggesting the involvement of carbohydrates in the binding reaction. The sulfated polysaccharides did not inhibit the binding of blotted gp37 and solubilized sperm components. In addition, blotted gp37 bound with several other nonsperm proteins, e.g., fetuin and transferrin, and cellitissue extracts, e.g., boar sperm and liver. The biological role of gp37 in sperm-envelope binding apparently lacks cell/molecular binding specificity and fertilization-relevant inhibition by sulfated polysaccharides. However, gp37 may play a role in fertilization “since only selected molecules (e.g. sperm membrane proteins and envelope components) would be proximal to each other in in situ interactions. Therefore, spatial or topological proximity may alleviate the necessity for molecular specificity” (Lindsay and Hedrick, 1988). It is clear from this incompletely satisfying conclusion that additional experiments using different methods and other anuran species are needed to identify VE component(s) functioning in sperm-envelope binding. Fertilization Envelope. The VE is converted to the FE at fertilization by the sperm- triggered release of the cortical granule contents. Three ultrastructural modifications of the ECM have been observed. Formation of a n electron-dense amorphous layer a t the interfacing surfaces of the VE and jelly coat layer J1 was first observed using X. Zaevis (see Fig. 6) (Grey et al., 1974). The presence of a n electron-dense layer on the outer surface of the FE of Rana temporaria eggs is apparent in micrographs published by Wartenberg and

Schmidt (1961, Abb. 5), but the authors did not comment on its presence. The amorphous fertilization or F layer of the FE is composed of two regions, a more electron-dense region closer to the envelope, F,, and a less electron-dense or dispersed region, Fd, located closer to J,. With the transmission electron microscopic methods used (glutaraldehyde and OsO, fixation, thin sections), no other ultrastructural modifications in the envelope or perivitelline space ECM are apparent. More recent studies using QDR methods have revealed two more ultrastructural changes when the VE is converted to the FE (Larabell and Chandler, 1988). The VE-derived envelope component of the FE, referred to as VE*, is restructured so that i t appears multilayered with fibrous sheets that twist and curl and sometimes merge. The sheets are interconnected by medium-sized (10 nm) and small-sized (4 nm) fibers. The structure was described a s reminiscent of the Greek pastry bachlava or a puff pastry. The large fibers that dominate the VE (19 nm) are apparently absent from the converted envelope. The loss of the predominant 19 nm fibers from the VE and the appearance of the predominant fibrous sheets in the VE” moiety of the FE suggest that the 19 n m fibers are converted into the fibrous sheets. A structural change also occurs in the perivitelline space a t fertilization. The H F layer, intimately associated with the VE of the unfertilized egg and with the tips of the egg microvilli intercalated into the layer, is converted into a layered sheet by the assembly of 36 nm particles onto its surface. The particles aggregate and then transform into a n exceedingly smooth surfaced layer (smooth for biological particles that are presumably protein or glycoprotein). The particles seem almost to “melt” or fuse together. The fibrous part of the S layer remains filamentously connected to the tips of the microvilli, whereas the FE has elevated away from the S layer and the cell surface. Thus the HF layer that was closely applied to the VE is far removed from the FE after the cortical reaction. Formation of the S layer apparently divides the perivitelline space into two compartments, one defined by the FE and the S layer and the other by the S layer and the plasma membrane of the cell. The S layer is produced by a combination of the HF layer and components from the cortical granules (Larabell and Chandler, 1989a). Treatment of dejellied oviposited eggs (possessing a n intact VE) with the contents of cortical granules produced a n in vitro-generated S layer that was structurally equivalent to that formed in vivo by the cortical reaction. The S layer is apparently destroyed by OsO, and ethanol dehydration, a fixative and condition previously used for transmission electron microscopy of thin sections. The sensitivity of the ECM structures to these conditions likely explains why they are not observed in transmission electron microscopy of thin sections. The FE from fertilized or ionophore A23187-activated eggs has been isolated after the eggs were dejellied using nylon screens. This was first accomplished using X . Zaevis (Gerton and Hedrick, 1986b; Wolf et al., 1976) and has also been done with B. arenarum (Miceli et al., 1977) and B.japonicus (Lindsay et al., 1988). The


ultrastructure of the isolated envelope as determined by transmission electron microscopic and light microscopic methods is equivalent to that of the in situ envelope. Three alterations in the macromolecular composition of the X . laevis VE occur when the VE is converted to the FE (Table 1). Two glycoproteins are added, one with a n MW in the range of 40K to 45K and the other with a large but undetermined MW. Two glycoproteins undergo limited hydrolysis with a loss of approximately 3K mass units (gp69,64 -+ gp66,61). These two processed glycoproteins are closely related and have the same or a very similar polypeptide chains but differ in their glycosylation. The hydrolytic processing apparently involves the C-terminal end of the polypeptides, as the N-terminal ends of all FE components are chemically blocked. C-terminal processing is also involved in the gp43 --+ gp41 reaction catalyzed by oviductin in the CE to VE conversion. Similar limited proteolytic changes, involving the conversion of gp65,61 to gp62,58 glycoprotein components, occur in the B . japonicus VE following fertilization or activation of eggs (Lindsay et al., 1988). The chemical changes in the X . laevis and B . japonicus VE are accompanied by marked physical changes in the envelope, in addition to the ultrastructural changes discussed above (Bakos et al., 1990a,b; Gerton and Hedrick, 1986a,b; Lindsay et al., 1988; Lindsay and Hedrick, 1989; Wolf e t al., 1976). Table 2 lists the physical changes that have been measured. The altered physical properties of the envelope are undoubtedly a reflection of the chemical alterations that include the addition of the F layer ( X . Zaeuis) and limited proteolysis of glycoproteins (both species). In addition to those changes listed in Table 2, conversion of the VE to the FE reduced the suseptibility of the envelope to hydrolysis by sperm lysins in B. arenarurn and B . japonicus (Cabada et al., 1989; Raisman and Barbieri, 1969; Yamasaki et al., 1988). Differences in the binding of concanavalin A have been reported for B. arenarurn CE, VE, and FE (del Pino and Cabada, 1987). Some of the factors responsible for the VE to FE conversion (the ultrastructural, physical, and chemical changes in the envelope) have been identified in X . laevis and B. japonicus (Yamasaki and Katagiri, 1989) and the molecular mechanisms involved in the conversion postulated for X . laevis (Larabell e t al., 1989b; Lindsay and Hedrick, 1989 and unpublished observations; Prody et al., 1985). The limited proteolysis of gp69,64 is apparently performed by a linked reaction of two proteases released by the cortical reaction. The cortical granule exudate recovered from dejellied, activated eggs catalyzes the conversion of gp69,64 to gp66,61. This chemical conversion reaction is not affected by the state of the gp43/41 component, as the exudate will catalyze the gp69,64 to gp66,61 reaction in either the CE or the VE. The exudate contains two proteases, one of which is arginyl peptide bond specific and the other phenylalanyl peptide bond specific, as determined using synthetic peptide substrates. The proteases have different pH optima (pH 9.0 and 7.5, respectively). The Arg-specific protease has a n MW of 45K and the Phe-specific protease a n MW of 30K (by the detection of proteolytic activity after SDS-PAGE

329

separation of cortical granule exudate macromolecules). Serine active site reagents and specific substrate affinity reagents inhibited the protease activities as well as the conversion of gp69,64 to gp66,61. Using the inhibitor information in addition to the apparent time-dependent activation of Phe-specific activity in the cortical granule exudate, a model was formulated wherein the Arg-specific protease was involved in activating the Phe-specific protease that in t u r n catalyzed the gp69,66 hydrolysis. I t is postulated that the gp69,64 to gp66,61 reaction is responsible for the physical changes (hardening) of the envelope. However, some experimental observations are not consistent with this postulate. The kinetics of the change in the physical properties of the envelope (hardening) in activated eggs correlated with the kinetics of cortical granule exocytosis (Greve e t al., 1985; Monk and Hedrick, 1986; Wolf et al., 1976), namely, with a 50%time of 7.3 min. Thus a factor in the cortical granules is responsible for envelope hardening. However, the 50% time for the gp69,64 hydrolysis was 13.5 min. This suggests that the proteolysis of gp69,64 is not responsible for the alteration of the physical properties of the envelope. Such a conclusion is puzzling, because hydrolysis of gp69,66 is the only chemical change thus far detected in the VE* component of the FE. An alternate possibility is that hydrolysis of gp69,64 does not have to go to completion in order to effect a change in the physical properties of the envelope. Perhaps hydrolysis of a few gp69,64 molecules can trigger a conformational change in some molecules that in t u r n induces changes throughout the entire envelope, thereby altering the envelope's gross physical properties with only limited alterations in envelope chemistry. This possibility is somewhat analogous to the allosteric control properties of some binding proteins and enzymes where binding of allosteric ligands alters the protein's conformation. Covalent modifications that control protein conformation have also been demonstrated. Alteration of the fibrous elements in the VE involve the apparent conversion of the relatively large 19 nm fibers into the fibrous sheets characteristic of the VE* mentioned previously. It appears that this ultrastructural alteration is triggered or caused by limited proteolysis of the gp69,64 components catalyzed by the Arg- andlor Phespecific proteases released in the cortical reaction. These changes are different in specific, but similar in general, to those occurring in the CE to VE conversion. The cellular location of the Arg- and Phe-specific proteases would seem to be the cortical granules, because the proteolytic activities are found in the cortical granule exudate. However, recent transmission electron microscopic cytochemical localization experiments, using Phe-containing peptide substrates whose enzyme-produced products are osmophillic, have localized the Phespecific protease activity in the perivitelline space fibers (Larabell et al., 1989b). This is the first specific molecule to be identified as existing in the perivitelline space ECM of any unfertilized animal egg. This surprising finding suggests that the appearance of the Phe-specific protease in the cortical granule exudate is due to its release from the perivitelline space ECM and its being "washed" through the envelope by a n exocy-

330


totic wave of cortical granule contents emanating from the cell surface. Perhaps its release from the perivitelline space involves activation of the enzyme by the presumably cortical granule-contained Arg-specific protease. However, the subcellular location of the Argspecific protease has yet to be determined. The molecular mechanism responsible for the formation of the F layer of the X. laevis FE was suggested to be a lectin-ligand-binding reaction (Wyrick et al., 1974). A similar suggestion was subsequently made for modification of the VE in B. arenarum but definitive evidence for the presence of a lectin-ligand reaction has yet to be published (Cabada et al., 1987). In X. laevis, a galactosyl-specific lectin is released from the cortical granules via exocytosis, diffuses through the VE, and binds to its ligand located on the outer face of the envelope (Greve and Hedrick, 1978). The ligand was originally proposed to be a component of the innermost jelly coat layer, because mercaptan-solubilized jelly possessed ligand activity (Wyrick e t al., 1974). It was subsequently shown by Yoshizaki and Katagiri (1984) and by Yoshizaki (1984) that a layer separate from the VE and the innermost jelly coat layer, J,, was applied to the egg as it passed through the PR-oviduct (see Fig. 5). The layer was named the prefertilization (PF)layer, because it was situated in the same location as the F layer. The PF layer is mercaptan soluble and is solubilized with the jelly coat layers, whereas the F layer is mercaptan insoluble. Polyclonal antibodies made to isolated PR secretory granules and purified by absorption reacted with the PR2 oviduct, the PF layer, and the F, portion of the F layer. They also reacted with EDTA-solubilized F layer from isolated FEs. The PF layer, then, is a secretory product of the PR2 oviduct and, in combination with the cortical granule lectin, forms the F layer. The molecules composing the PF layer have yet to be isolated and their ligand activity for cortical granule lectin demonstrated. Treatment of jellied oviposited eggs with purified cortical granule lectin produces precipitin lines of reaction with the PF layer and also with the innermost jelly coat layer, J, (Nishihara and Hedrick, unpublished observations). In addition, using antibodies to mercaptan-solubilized jelly coat layers and immunoelectrophoretic methods, three molecules have been identified as having ligand activity for the cortical granule lectin (Birr, 1979; Birr and Hedrick, submitted for publication). Although we can conclude from the above experiments that the PF layer contributes to the formation of the F layer, the molecules in the egg ECM (the total ECM or egg integuments, from the fibers of the perivitelline space to the outermost jelly coat layer) that are ligands for the cortical granlule lectin need to be identified and isolated and their structure-function properties determined to understand fully the mechanism and role of lectin-ligand interactions in regulating sperm-egg inter actions. Several different experiments can be cited that support the lectin hypothesis for the formation of the F layer. These include purification of a galactosyl-specific lectin from the cortical granule exudate (Chamow and Hedrick, 1986; Nishihara et al., 1986; Yoshizaki, 19861,immunocytochemical studies showing the corti-

Fig. 7. TEM micrograph of the F material precipitated by the addition of Ca2 ' . The material was prepared as described in Materials and Methods. Bar = 0.05 pm; x 160,000.

cal granule lectin located in the cortical granules before fertilization and the perivitelline space and FE (VE" and F layer) after fertilization (Greve and Hedrick, 1978; Yoshizaki, 1989), and, in the SDS-PAGE analysis of the FE macromolecules, the two components added to the VE corresponding to the cortical granule lectin (subunit MW of 40-45K) and the cortical granule lectin ligand (large undetermined MW component; Nishihara et al., 1983). An additional experiment that supports the lectin hypothesis for the formation of the F layer is the following reconstitution experiment. Isolated FEs were separated into solubilized F layer material and particulate VE" by extracting the envelopes with Gal or EDTA. The dialyzed soluble F material contained the cortical granule lectin, a s shown by immuno-doublediffusion (Ouchterlony) experiments using antibodies against the purified cortical granule lectin (precipitin lines of identity were obtained between the F material and purified cortical granule lectin) and by SDS-PAGE analysis (the F material contained bands identical to purified cortical granule lectin). When CaZ' was added to the solution of F material, a precipitate formed that, when viewed with the transmission electron microscope, showed a n amorphous structure that contained areas corresponding to the F, and F, regions of the in situ F layer (Fig. 7). The VE* was cleanly stripped of its F layer and had a n appearance equivalent to the VE moiety of the FE (Fig. 8). When the soluble F material was mixed with the isolated VE" and Ca2+ added, the F layer was reformed as a single layer on the VE* (Fig. 9). As can be seen, the reconstituted FE had a n F layer that was structurally imperfect compared with the in situ formed F layer. This is not surprising because addition of the interactinglprecipitating lectin-ligandCa2+ complex could not be as regular and ordered as that assembled from the cortical granule lectin re-


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Fig. 8. TEM micrograph of the isolated FE and the VE*. The VE* (right)was prepared from the FE (left) as described in Materials and Methods. Bar = 0.2 pm; ~ 5 2 , 0 0 0 .

leased in cortical grctnule exocytosis. Thus the reconstitution of the FE from its isolated component parts also supports the lectin hypothesis for the formation of the F layer. In the reconstitution experiments, formation of the F layer on a single side of the envelope in situ is explained by the deposition of the PF layer only on the outer surface of the envelope. Cortical granule lectin diffusing through the perivitelline space and the envelope would subsequently form a precipitin F layer when it binds to its ligand in the PF layer located only on the outer surface of the envelope. Thus the asymmetry of the in situ-generated FE can be explained by the one-sided distribution of the PF layer. However, in the reconstitution experiments, the asymmetry of the reconstituted FE cannot be explained by the location of the PF layer, since the PF layer does not exist as a preformed structural entity associated with the envelope. The asymmetry of the reconstituted FE requires that the cortical granule lectin, the ligand, or the lectin-ligand complex specifically interact (bind) with glycoproteins asymmetrically located on the outer surface of the envelope. In addition, it is clear that some binding interaction between the F layer and VE* must occur, as the F layer is not lost in the vigorous washing procedures used to isolate the FE. The results of surface radiolabeling experiments using isolated and in situ envelopes suggested that all envelope components were located on the outer and inner surfaces. However,

there was a change in accessibility for [l"I]-radiolabeling of envelope glycoproteins when the VE was converted to the FE (Nishihara et al., 1983). These results demonstrate that there is no uniquely located envelope component that could serve to bind the reconstituted F layer asymmetrically to the VE*. However, the radiolabeling experiments only measured the topological distribution of the Tyr residues in the envelope glycoproteins. The envelope glycoproteins are known to be microheterogeneous because of their carbohydrate moieties a s shown by 2D-PAGE analysis (Gerton and Hedrick, 1986a,b). Nothing is known about the topological distribution of the carbohydrate moieties of the envelope glycoproteins, so it is possible that the lectin or the lectin-ligand complex additionally binds to glycoprotein oligosaccharide side chains that are uniquely or predominantly located on the outer surface of the envelope. The envelope glycoprotein(s) that bind the F layer to the VE* need to be identified. An additional experiment using isolated VE instead of VE* was undertaken to examine further the asymmetric binding of the F layer. Similar to the experiment described above (Fig. 9), Ca2+ was added to a mixture of soluble F material and particulate VE (in place of VE*) and the resulting envelope observed by transmission electron microscopy. Surprisingly, two F layers were found attached to both surfaces of the VE (Fig. 10). Two explanations can be offered for this observation.

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~ i 9. ~ TEM , micrograph of the reconstituted fertilization enve. lope. Soluble F material was mixed with the VE* in the presence of Ca'- as described in Materials and Methods. Bar = 0.2 km; x 52,000.

1. The F layer adhesion to the two VE surfaces involves separate structuresimacromolecules in the envelope. For instance, if the H F layer were attached to the inner face of the isolated VE and it interacted with the F layer, this could be a macromolecularly different interaction than that which occurs on the outer surface of the envelope. The HF layer is ultrastructurally different from the outer surface of the VE in terms of its fiber content and organization, although nothing can be said about the differences or similarities a t the molecular level. The H F layer is converted to the S layer by binding or assembly to its net-like structure of 36 nm particles derived from the cortical granules. The cortical granule lectin is the major constituent of the cortical granules (Nishihara et al., 1986). By aggregating approximately 16 cortical granule lectin molecules, 36 nm particles can be constructed that geometrically correspond to those forming the S layer. After its formation, the S layer remains attached to the tips of the microvilli and is separated from the envelope a s the FE osmotically lifts off and moves away from the S layer and the cell surface. After the envelope elevates, the fibers of the H F layer are no longer atached to the envelope (VE*) and the inner face of the envelope could not interact with a lectin or lectin-ligand complex. The binding of the lectin or lectin-ligand complex to the

Fig. 10. TEM micrograph of the reconstituted envelope obtained from VE and F material. Souble F material was mixed with the VE in the presence of Ca2' as described in Materials and Methods. Bar = 0.2 Fm; 64,000,

outer surface of the envelope is undisturbed relative to that taking place a t the inner surface, and hence an asymmetric, reconstituted FE would be produced. 2. The VE may have a n equivalent potential for binding F material to both of its surfaces, but the VE to VE* conversion molecularly alters the inner aspect of the envelope so i t can no longer interact with F material. For instance, if the gp69,64 components, which are limit hydrolyzed in the VE to FE conversion, were protected from proteolysis when the lectin-ligand complex (but not the lectin by itself) bound to them, proteolysis of these components on the inner aspect of the VE during cortical granule exocytosis would produce a n asymmetric distribution of the gp69,64 components in the resulting VE*. This ordered binding process could give rise to a symmetrical binding of the F material with the VE and a n asymmetrical binding with the VE*. The gp69,64 components are unequally distributed in the VE, as shown by radiolabeling experiments (more on the outer surface; Nishihara e t al., 1983). Thus the VE to VE* conversion could increase this unequal distribution if the F material protected the gp69,64 components from proteolysis. Experiments designed to measure the binding of F material by the H F layer (attached to the VE or attached to the microvilli of the naked egg [envelope removed], depending on how the H F layer partitions when the VE is removed) and to

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determine the topographical distribution of envelope molecules e.g., gp69,64 and their carbohydrate and protein moieties, using specific antibodies with known epitope (protein and carbohydrate) specificities, could distinguish between these two possible explanations. In addition, experiments t o determine the binding of F material to the isolated CE could be instructive.

CONCLUDING REMARKS To increase our understanding of the structure-function properties of the anuran egg ECM, experiments in the future need to be focused a t three different organizational levels. At the molecular level, analysis of the macromolecular composition of the ECM substructures needs to be completed, i.e., individual jelly coat layers and the organization of macromolecules within an individual layer; egg envelopes and their substructures, such as the 70-100 nm fibrous bundles; perivitelline space structures, such as the HF layer. The analytical phase of anuran egg ECM research has provided a basis for synthetic and integrative hypothesizing as to the structure-function relations of the ECM, but the information base is incomplete and insufficient. The chemical structures of the macromolecules that modify and alter the ECM need to be determined. For instance, the proteolytic processing of envelope components by proteases released in cortical granule exocytosis and the cortical granule lectin-PF layer ligand-binding reaction need to be defined at the chemical level to provide an understanding of structure-function relations a t the supramolecular level. Recombinant DNA methods (elegant in their apparent simplicity, appealing, and powerful) will greatly assist progress in this regard, but the application of contemporary physicochemical methods (classical, less appealing, but powerful none the less) are also required because recombinant DNA methods cannot yet assist in providing primary structure information on the oligosaccharide moieties of glycoproteins, the major constituents of the anuran egg ECM. Fortunately, major improvements in methods available for determining carbohydrate structures have occurred during the last decade, but substantial amounts of material are still required for analysis. In this respect, anurans may continue to be particularly favorable systems for study, because relatively large amounts of gametes are readily obtainable compared with other animals, e.g., mammals. The utility of anurans in reproductive biology research (which includes developmental biology) is, of course, not a new perception, as is apparent from even a brief reading of the history of reproductive biology. At the supramolecular level, the individual macromolecules of the ECM are assembled and organized into higher order structures and revealed by ultrastructural methods as startlingly beautiful, complex molecular ensembles. The dazzling architecture of these structures must be understood in terms of their construction and the organization of their component parts to comprehend how they function. Electron microscopy is one of the techniques that can transcend the molecular and cellular levels of biological organization to provide the information needed. The continued merging of techniques, e.g., immunocytochemical and

QDR methods, will be very revealing as they are applied to the anuran egg ECM. The integration of the molecular and subcellular structural information with biological function at the cellular level (sperm-egg interactions) into encompassing hypotheses is the ultimate goal. In this regard, a comparative approach will be of great assistance in unveiling the fundamental properties of the egg ECM. The number of hypotheses concerning the egg ECM (anuran or other) that incorporate structure-function information at all the relevant levels of cellular organization, transcending molecules and cells, are few. Our limited success in this regard should be an encouragement to continue to search and a reminder that our understanding of the structure-function properties of the egg ECM is very incomplete.

ACKNOWLEDGMENTS This review is dedicated by J.L.H. to his past and present students for the pleasure of their partnership in research on X . laevis and to the later author of “Chemistry and Physiology of Fertilization,” Albert0 Monroy, on the 25th year anniversary of his book‘s publication date. He gratefully acknowledges the significance of Monroy’s book in his introduction to fertilization research. The excellent assistance of Robert J. Munn and Heather A. Fabry in preparing the electron micrographs and of Heather A. Fabry for the quantitative analysis of envelope compositions is gratefully recognized. Partial support for J.L.H was provided by the Education Abroad-Faculty Exchange Program of the University of California during the writing of this article. He further thanks Professor Chiaki Katagiri, Hokkaido University, for providing writing and laboratory facilities, for many helpful discussions, and for the joy of his friendship. The experimental work reported here in the senior author’s laboratory was generously supported in part by USPHS research grant HD04906. REFERENCES Bakos, M., Kurosky, A,, and Hedrick, J.L. (1990a) Physicochemical characterization of progressive changes in the Xenopus laeuis egg envelope following oviductal transport and fertilization. Biochemistry, 29:609-615. Bakos, M., Kurosky, A., and Hedrick, J.L. (1990b) Enzymatic and envelope converting activities of pars recta oviductal fluid from Xenopus laeuis. Dev. Biol., 138:169-176. Balinsky, B.I. (1966) Changes in the ultrastructure of amphibian eggs following fertilization. Acta Embryol. Morphol. Exp., 9:132-154. Birr, C.A. (1979) Immunoelectrophoretic Studies of the Jelly Coat Ligand for the Cortical Granule Lectin of Xenopus laeuis Eggs. Ph.D. Thesis, University of California, Davis. Cabada, M.O., Manes, M.E., and Gomez, M.I. (1989) Spermatolysins in Bufo arenarum: Their activity on oocyte surface. J . Exp. Zool., 249229-234. Cabada, M.O., Mariano, M.I., and Raisman, J.S. (1978) Effect of trypsin inhibitors and concanavalin A on the fertilization of Bufo arenarum coelomic oocytes. J. Exp. Zool., 204:409-416. Cabada, M.O., Mariano, M.I., and Raisman, J.S. (1987) Cortical granules products and fertility prevention in Bufo arenarum oocytes. J. Exp. Zool., 241:359-367. Chamow, S.M., and Hedrick, J.L. (1986) Subunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus Zueuis eggs. FEBS Lett. 206:353-357. del Pino, E.J., and Cabada, M.O. (1987) Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization. Gamete Res., 17:333-342.

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Katagiri, C. (1986) The role of oviducal secretions in mediating gamete fusion in the toad, Bufo bufojaponicus. Adv. Exp. Med. Biol., 207:151-166. Katagiri, C. (1987) Role of oviducal secretions in mediating gamete fusion in anuran amphibians. Zool. Sci., 4:l-14. Katagiri, C., Iwao, Y., and Yoshizaki, N. (1982) Participation of oviducal pars recta secretions in inducing the acrosome reaction and release of vitelline coat lysin in fertilizing toad sperm. Dev. Biol., 94:l-10. Kobayashi, H. (1954) Hatching mechanism in the toad, Bufo uulgaris formosus. 1. Observations and some experiments on perforation of gelatinous envelope and hatching. J . Fac. Sci. Univ. Tokyo (Zool.), 7:79-88. Larabell, C.A., and Chandler, D.E. (1988) The extracellular matrix of Xenopus laeuis eggs: A quick-freeze, deep-etch analysis of its modification at fertilization. J . Cell Biol., 107:731-741. Larabell, C.A., and Chandler, D.E. (1989a) In vitro formation of the “ S ’ layer, a unique component of the fertilization envelope in Xenopus laeuis eggs. Dev. Biol., 130:356-364. Larabell, C.A., and Chandler, D.E. (198913) The coelomic envelope of Xenopus laeuis eggs: A quick-freeze, deep-etch analysis. Dev. Biol., 131:126-135. Larabell. C.A.. Hardv. D.M.. and Hedrick. J.L. (1989a) Fibers of the coelomic envelope bf Xenobus laeuis eggs are unbundled by a n oviductal enzyme. J . Cell Biol., 109:128a. Larabell, C.A., Lindsay, L.L., and Hedrick, J.L. (198913) Localization of a chymotrypsin-like enzyme to the perivitelline space of Xenopus laevzs eggs. J . Cell Biol., 109:128a. Lillie, F.R. (1919) Problems of Fertilization. The University of Chicago Press, Chicago, p. 31. Lindsay, L.L., and Hedrick, J.L. (1988) Identification of Xenopus laeuis sperm and egg envelope binding components on nitrocellulose membranes. J . Exp. Zool., 245286-293. Lindsay, L.L., and Hedrick, J.L. (1989) Proteases released from Xenopus laeuis eggs at activation and their role in envelope conversion. Dev. Biol., 135202-211. Lindsay, L.L., Yamasaki, H., Hedrick, J.L., and Katagiri, C. (1988) Egg envelope conversion following fertilization in Bufo japonicus. Dev. Biol., 130:37-44. Mariano, M.I., de Martin, M.G., and Pisano, A. (1984) Morphological modifications of oocyte vitelline envelope from Bufo arenarum during different functional states. Dev. Growth Differ., 26:33-42. Metz, C.B. (1967) Gamete surface components and their role in fertilization. In: Fertilization, Vol. 1. C.B. Metz and A. Monroy, eds. Academic Press, New York, pp. 163-236. Miceli, D. (1986) Oviducal pars recta as factor in fertilization properties and hormonal regulation in the toad Bufo japonicus. Adv. Exp. Med. Biol., 207:167-183. Miceli, D., Fernandez, S.N., Raisman, J.S., andBarbieri, D.F. (1978a) A trypsin-like oviducal proteinase involved in Bufo arenarum fertilization. J. Embryol. Exp. Morphol., 48:79-91. Miceli, D.C., del Pino, E.J., Barbieri, F.D., Mariano, M.I., and Raisman, J.S. (1977) The vitelline envelope to fertilization envelope transformation in the toad Bufo arenarum. Dev. Biol., 59:lOl-110. Miceli, D.C., Fernandez, S.N., and del Pino, E. (197813) An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes. Biochim. Biophys. Acta, 526:289-292. Monk, B., and Hedrick, J.L. (1986) The cortical reaction in Xenopus laeuis eggs: Cortical granule lectin release as determined by radioimmunoassay. Zool. Sci., 3:459-466. Newport, G. (1851) On the impregnation of the ovum in the amphibia. Philos. Trans. R. SOC.Lond. (First Series; Part 11, 141:169-242. Nishihara, T., Gerton, G.L., and Hedrick, J.L. (1983) Radioiodination studies of the envelopes fromxenopus laeuis eggs. J. Cell. Biochem., 22:235-244. Nishihara, T., Wyrick, R.E., Working, P.K., Chen, Y., and Hedrick, J.L. (1986) Isolation and characterization of a lectin from the cortical granules of Xenopus laeuis eggs. Biochemistry, 25:6013-6020. Prody, G.A., Greve, L.C., and Hedrick, J.L. (1985) Purification and characterization of a n N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laeuis eggs. J . Exp. Zool., 235:335-340. Raisman, J.S., and Barbieri, F.D. (1969) Lytic effects of sperm suspensions on the vitelline membrane ofBufo arenarum oocytes. Acta Embryol. Exp., 1:17-26. Raisman, J.S., and Cabada, M.O. (1977) Acrosome reaction and proteolytic activity in the spermatozoa of a n anuran amphibian, Leptodactylus chaquensis. Dev. Growth Differ., 19:227-232.

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