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Chapter 12 Isolation of Extracellular Matrix Structures from Xenopus laevis Oocytes, Eggs, and Embryos
Chapter 12
Isolation of Extracellular Matrix Structures from Xenopus laevis Oocytes, Eggs, and Embryos JERRY L. HEDRICK Department of Biochemistry and Biophysics University of California at Davis Davis, CaIifornia 35616
DANIEL M. HARDY Howard Hughes Medical Institute University of Texas Southwestem Medical Center Dallas, Texas 75235
I. Introduction 11. Methods
A. B. C. D. E.
Induction of Ovulation and Obtaining Oviposited Eggs Preparation of Egg Jelly Coat Layers Sieving Methods for Isolation of Egg Envelopes Obtaining Oviposited Eggs for Vitelline Envelope Preparation Obtaining Activated or Fertilized Eggs for Fertilization Envelope Preparation F. Obtaining Coelomic Eggs for Coelomic Envelope Preparation G. Obtaining Oocytes for Ovarian Envelope Preparation H. Radioiodination of Isolated Envelopes 111. Discussion A. Jelly Coat Layers B. Envelopes References
23 1 METHODS IN CELL BIOLOGY. VOL. 36
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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I. Introduction The extracellular matrix (ECM) surrounding amphibian eggs is composed of jelly coat layers, the egg envelope, and the fibrous elements of the perivitelline space (Fig. 2). The biological functions of the egg ECM in fertilization and development were first noted more than a century ago when Newport (1851) reported that unjellied eggs recovered from the coelom were not fertilizable. We now appreciate that the oviductally produced jelly coat layers of amphibian eggs are required for the process of sperm capacitation and/or induction of the sperm acrosome reaction (for review, see Hedrick and Nishihara, 1991; Katagiri, 1987). The oviposited egg envelope provides an initially penetrable structure to the sperm which is subsequently modified by products released in the cortical reaction to a sperm-impenetrable structure in the block to polyspermy reaction (for reviews, see Schmell et al., 1983; Elinson, 1986). However, the ovulated egg recovered from the coelom possesses an envelope which is also impenetrable to sperm as originally shown in Rana pipiens and Bufo japonicus (Elinson, 1973; Katagiri, 1974). Thus, the egg envelope goes from a sperm-impenetrable state (the coelomic egg) to a penetrable state (the oviposited egg) and back to an impenetrable state (the fertilized egg or zygote). At fertilization the egg envelope is chemically modified and its macromolecular permeability properties changed (discussed in Schmell et al., 1983). This permeability change (hardening) results in an osmotically driven envelope elevation, owing to the influx of water into the perivitelline space. The jelly after fertilization functions as a “sticky substrate” for the adherence of the zygote to objects in its surroundings, protects the zygote against physical damage, and also provides a microbiological barrier (bacteria are rarely found within the jelly coat layers). Thus, after fertilization the ECM protects the developing embryo and functions as a barrier for the chemical and biological regulation of the embryo environment. This protective function of the ECM persists until the embryo develops into a free swimming tadpole and hatches from the ECM. The hatching process involves both physical and enzymatic mechanisms (Carroll and Hedrick, 1974).The ECM of the egg and the embryo, therefore, play significant roles in fertilization and development. Contemporary approaches to the structure-function relations of the ECM and its component macromolecules using Xenopus laeuis have utilized electron microscopic, biochemical, and immunological methods (for reviews, see Hedrick and Nishihara, 1991; Larabell and Chandler, 1991). The macromolecular compositions of the jelly coat layers (Table I) and the various forms of the egg envelopes have been determined (Fig. 1). Isolation of the individual glycoproteins composing the envelopes is currently in progress and
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TABLE I GLYCOPROTEIN COMPOSITION OF Xenopus laeuis JELLY COAT LAYERS'
Number of different glycoproteins Jelly coat layer Jl
J, J3
Total
Electrophoresis ID
SVC
IM
CA
SDS-A
2 2
2 2 4 8
3 2 4 9
2 2 5 9
3 2
4
8*
4 9'
a Abbreviations: ID, immunodiffusion; SVC, sedimentation velocity centrifugation; IM, immuno; CA, cellulose acetate; SDS-A, sodium dodecyl sulfate-agarose. Adapted from Yurewicz et al., 1915. One additional component common to J, and J3 was detected. 'Two additional components common to J , and J, were detected.
is a necessary prerequisite to the use of recombinant DNA methods for determination of the structure-function relations of the ECM glycoproteins (Gerton et al., 1982; Nishihara et al., 1986; Lindsay and Hedrick, 1988).
11. Methods
A. Induction of Ovulation and Obtaining Oviposited Eggs The quality and quantity of eggs and the timing of ovulation are greatly improved by using a pregnant mare serum gonadotropin (PMSG) priminghuman chorionic gonadotropin (HCG) ovulating protocol over that which uses HCG alone (Hedrick and Nishihara, 1991). Accordingly, females kept on a 12 hours light/l2 hours dark schedule at 20°-23"C are injected into the dorsal lymph sac with 35 IU of PMSG dissolved in 1 ml of 0.15 M NaCl. After 96 hours, ovulation is induced by the injection into the dorsal lymph sac of 500-1000 IU of HCG. After 5-6 hours, eggs are stripped from the females into DeBoers solution. DeBoers solution is 110 mM NaCl, 1.3 mM CaCl,, and 1.3 mM KCl, adjusted to pH 7.2-7.8 with NaHCO, (Katagiri, 1961).Normally, three or four strippings are done at 1.5- to 2-hour intervals and 1500-3000 eggs per female collected. Females are returned to their tanks and rested for at least 6 weeks before they are used again for egg production.
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B. Preparation of Egg Jelly Coat Layers 1.
COLLECTION OF INDIVIDUAL JELLYCOAT LAYERS
Individual jelly coat layers can be microdissected from DeBoers-washed eggs using a pair of sharp watchmaker’s forceps and a dissecting microscope (Yurewicz et al., 1975). A slight hydration of the jelly coat layers in DeBoers solution aids in their removal. This is usually accomplished by allowing the oviposited eggs to sit in DeBoers solution for 20-30 minutes at room temperature, The use of dilute salt solutions in this procedure is not possible as jelly coat layer J, becomes so sticky that manipulation of the eggs is difficult. However, for the recovery of eggs with only J, attached, this stickiness can be a benefit as the eggs adhere to the bottom of a culture dish and do not otherwise have to be held in place. The soft outermost layer, J3, can be cleanly removed from the tougher and more leatherlike J2 layer by gently stripping away J3 with one pair of forceps while using a second pair to hold the egg. Isolation of J2 requires piercing layer J2 with one pair of forceps and then teasing the torn J2layer away from the soft and highly hydrated J, layer with a second pair of forceps. J2 is invariably dissected with small amounts of adhering J, . Because of its soft, sticky “chewing gum” nature, J1 is most easily separated from the egg by solubilization in mercaptan solutions. Accordingly, J 3 - and J,-less eggs are immersed in 100 mM NaC1-50 mM Tris-HC1, pH 8.0, containing 2-5 mM dithiothreitol. The eggs are gently swirled, and dejellying is usually complete in 5-lo minutes at 22°C (as observed with the naked eye or with a dissecting microscope). The egg and vitelline envelope (VE) remain intact during this period. However, continued exposure to the dithiothreitol will dissolve the VE and lyse the egg. Decant the solution from the eggs and adjust the pH of the jelly solution to 6.5-7.0 for storage (oxidation of the sulfhydryl groups to disulfides occurs very slowly at neutral or acidic pH values; for long-term storage, 1 mM EDTA should also be added). A variety of mercaptans, such as mercaptoethanol, can be used instead of dithiothreitol, and DeBoers or other salt solutions can replace the NaC1-Tris solution. It should be noted that the rate of jelly dissolution is a function of the solution pH and mercaptan concentration (Gusseck and Hedrick, 1971). Lower pH values will slow the rate, and higher pH values will increase the rate of the dissolution reaction. The isolated insoluble jelly coat layers 5, and J2 can be washed in appropriate salt solutions and then dissolved in dithiothreitol solutions as was done with J, . The solubilized jelly coat layers are centrifuged at 12,000 g to remove particulate matter and dialyzed versus an appropriate buffer to adjust the pH and ion concentration. The jelly from a single egg contains
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approximately 41 pg of glycoprotein, with J , , and J , , and J, contributing 19, 5, and 17 pg, respectively. 2.
TOTALSOLUBILIZATION
OF
JELLY
For the preparation of jelly coat glycoproteins from all the jelly coat layers or for the preparation of dejellied eggs, oviposited eggs can be treated with mercaptan solutions. Place a monolayer of washed eggs in a flat-bottomed dish such as a crystallization dish (e.g., 20-30 ml of eggs in a 125 x 65 mm dish). Add 80 ml of 10 mM Tris-DeBoers solution adjusted to pH 8.9 and containing 45 mM mercaptoethanol and gently rock the dish. Dissolution of the jelly coat layers can be followed by the naked eye and should be complete in 3-5 minutes. As stated previously, continued exposure of the eggs to the mercaptoethanol solution will dissolve the VE and lyse the eggs. Decant the solubilized jelly and adjust the pH to 6.5-7.0. If the eggs are also to be recovered, rinse them several times in 10 mM Tris-DeBoers solution at pH 7.0-7.8. From 1000 eggs, approximately 41 mg of glycoprotein is recovered with a composition of 39% protein and 61 % carbohydrate. 3.
PREPARATION OF
35 S-LABELED
JELLY
The egg jelly contains sulfate esters with the majority, if not all, of the sulfate located in jelly coat layer J, (Yurewicz et al., 1975). [35S]Sulfate is readily incorporated into the J , jelly coat glycoproteins (Hedrick et al., 1974). Injection of 1-5 mCi into the dorsal lymph sac along with the HCG gives maximum incorporation into the egg ECM. Approximately 0.1% of the injected dose is incorporated with 3000-4500cpm/egg of which 42% is associated with the jelly and 58% with the egg. The jelly contains both free and glycoprotein-bound [35S]sulfate with the distribution ranging from 76 and 24%, respectively, from eggs collected in the first stripping to 33 and 67%, respectively, from eggs collected in the final stripping. The specific activity of thejelly is also a function of the stripping time, with the highest specific activity obtained in the last stripping. A second ovulation of the female 5-8 days later with an additional 2-5 mCi of [35S]sulfate will give smaller numbers of eggs but with higher specific activity of the jelly glycoprotein. Some 93% of the [35S]sulfate counts are incorporated into a single acidic glycoprotein in jelly coat layer J,.
C. Sieving Methods for Isolation of Egg Envelopes All forms of the egg envelope [VE, fertilization envelope (FE), coelomic envelope (CE), and ovarian envelope (OE)] are isolated using a common
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sieving method (Wolf et al., 1976).Jellyless eggs are poured into a large syringe (10-100 ml depending on the number of eggs used) and lysed by passage through an 18-gauge needle. The lysate is filtered through a 210-pm nylon screen suspended on the top of a wide-mouth bottle. Envelopes resting on the top of the screen are gently washed through the screen with a wash bottle containing ice-cold distilled water or 10 mM Tris-DeBoers solution, pH 7.0. This step removes the larger debris. The filtered envelope suspension is then poured through a 105-pm screen and the smaller particulate matter is washed through as with the above procedure. This sieving process is repeated (usually twice) until the envelopes are visually free from contaminating particulate material. The purified envelopes are washed from the screen into a conical centrifuge tube and collected by a 1- to 2-minute centrifugation in a clinical centrifuge (room temperature, 1500 9). The major envelope contaminant is yolk granules, which are solubilized by overnight storage of the envelopes at 4°C in a salt solution (2 M NaCl, 0.2 M imidazole-HC1,2mM CaCl, , pH 7.0; Lindsay and Hedrick, 1989). The particulate envelopes are collected by centrifugation and washed with ice-cold water, DeBoers solution, or 0.15 M NaCl, depending on the subsequent use of the envelopes. The envelopes are stored at 4"C, pH 7.0, in high salt solutions (0.1 M NaCl) or CaCl, solutions to prevent swelling of the envelopes and solubilization of envelope glycoproteins, particularly the gp57 component of the VE and the FE (Bakos et d., 1990a). Envelopes can be solubilized by heating for 10 minutes at 80°C in water adjusted to pH 9 with Tris or NaOH. The envelopes remain soluble when the solution pH is subsequently lowered to pH 7 with dilute HCl or acetic acid.
D. Obtaining Oviposited Eggs for Vitelline Envelope Preparation Oviposited eggs are collected using the hormonal stimulation methods stated above. The oviposited eggs are dejellied, lysed, and VEs collected by the sieving methods described above. Approximately 1 mg of glycoprotein (90% protein, 10% carbohydrate) is obtained per 1000 eggs. The VE is composed of seven glycoproteins as indicated in Fig. 1.
E. Obtaining Activated or Fertilized Eggs for Fertilization Envelope Preparation Jellied oviposited eggs are activated by adding the calcium ionophore A23187 (2-5 p M final concentration) in 0.05 DeBoers or 0.05 TrisDeBoers solution (Monk and Hedrick, 1986).The calcium ionophore A23187
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Envelope Glycoprotein FIG. 1. The glycoprotein composition of egg envelopes. The glycoproteins are noted on the abcissa without the prefix gp. For graphing reasons (log of the weight %), 0% is equivalent to 0.01%. (The glycoprotein composition of CE is from Gerton and Hedrick, 1986b;that of VE and FE is from Gerton and Hedrick, 1986a; quantification by image analysis is from Hedrick and Nishihara, 1991.)The glycoprotein composition of the OE is qualitatively the same as the CE (data not shown).
stock solution (2-5 mM) is prepared by dissolving the ionophore in absolute ethanol or dimethyl sulfoxide. The activated eggs are gently swirled at room temperature, and after 20 minutes the medium is decanted. The activated eggs are washed, dejellied, lysed, and the activated FEs collected by sieving. Approximately 1.3 mg of glycoprotein (88% protein, 12% carbohydrate) is obtained per 1000 eggs (Wolf et d.,1976).The FE is composed of at least nine glycoproteins as indicated in Fig. 1. An alternative method for preparing the FE is to fertilize eggs (Wolf and Hedrick, 1971).A single testis is macerated in a conical centrifuge tube with a glass rod in 1 ml of DeBoers solution (the sperm concentration is approximately 1 x lo7 cells/ml). DeBoers solution-washed eggs are spread in a monolayer in the bottom of a dish. The solution is decanted and replaced with 0.05 DeBoers or 0.05 Tris-DeBoers solution. Sperm are immediately
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added to a final concentration of at least 1 x 105/ml. The suspension is gently swirled, and, after 20 minutes at room temperature, the overlying solution is decanted, the eggs dejellied and lysed, and FEs collected by sieving. The fertilization layer can be removed from the FE by solubilization to obtain the activated egg envelope moiety, VE* (the vitelline envelope component of the FE). Isolated FEs are suspended in 0.5 M galactose, 1.0 mM CaCl,, and 10 mM Tris-HC1, pH 7.8, for 2 hours at room temperature (Nishihara et al., 1983). Alternatively, the fertilization layer can be solubilized with 5 mM EDTA, 10 mM Tris-HCI, 154 m M NaCI, pH 7.8 (Hedrick and Nishihara, 1991). The VE* is recovered by centrifugation (5000 g for 5 minutes), extracted an additional time with the galactose- or EDTAcontaining solution, and finally washed by centrifugation in ice-cold water or a buffer solution. The extract contains the solubilized fertilization layer components, namely, the cortical granule lectin and its ligand (Nishihara et al., 1986).
F. Obtaining Coelomic Eggs for Coelomic Envelope Preparation To obtain coelomic eggs, the oviducts are surgically ligated prior to the induction of ovulation (Bakos et al., 1990b). A female frog is anesthetized by immersion in 1 liter of MS222 (1.6 g of ethyl 3-aminobenzoate methane sulfonate/liter) at 0°C for 15-20 minutes. Arrange the frog on a cheeseclothcovered tray of ice (Fig. 3). By palpation, locate the posterior dorsal point of the scapula and make a 1-cm longitudinal incision through the skin about 1.5 cm posterior to the scapula and midway between the scapula and the lateral line (Fig. 3). The incision is best made by holding the skin taut with the fingertips and cutting with a scalpel. After cutting through the skin, the underlying muscle layers are lifted with forceps and cut with a pair of scissors to avoid cutting the underlying lung. There are three muscle layers, with the inner layer being very thin; cutting through it will expose the lung (Fig. 4). While holding the inner muscle layer with forceps, with a second pair of forceps, grasp the innermost muscle layer inside the incision in a dorsal direction, gently pull the muscle externally, and transfer the grasp of the second forcep to a pronged hemostat. This procedure will prevent the inner muscle layer from retracting into the incision. Gently pull the inner muscle layer through the incision (evert) little by little until the attachment of the oviduct to the muscle layer and the ostium of the oviduct is located. The pars recta oviduct is easily differentiated from the pars convoluta oviduct as the pars recta is heavily vascularized relative to the white, opaque pars convoluta (Fig. 5).
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FIG.2. Diagram of the extracellular matrix of the oviposited Xenopus laeuis egg. The ECM structures in the diagram are not to scale. CG, Cortical granules; PS, perivitelline space; VE, vitelline envelope; PL, prefertilization layer; J,, J,, J 3 , individual jelly coat layers. Fia. 3. Location of the incision site for oviduct ligation.
FIG.4. Exposure of the pleuroperitoneal cavity. The three muscle layers have been cut and parted to expose the underlying lung. Fic. 5. The pars recta and pars convoluta oviduct. The pars recta is heavily vascularized compared to the pars convoluta (forceps at left). The pars recta is attached to the body wall (forceps at top) and leads to the ostium.
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FIG.6. Ligating the oviduct. FIG.7. The sutured incision at the completion of the ligation procedure.
Use 5-0 silk suture or single-use hemostatic silver clips to ligate the oviduct (Fig. 6). It is important to ligate the oviduct as close to the ostium as possible. Release the pronged hemostat and tuck the ligated oviduct back into the body cavity by lifting the body wall with forceps. Suture the three muscle layers of the body wall with 4-0 gut or close the incision with surgical staples. The three layers do not have to be separately sutured, but they must be well sutured to prevent leakage of ovulated eggs through the incision. Suture the skin with 3-0 silk suture (Fig. 7). If the frog begins to recover prematurely from the anesthesia, place some flakes of ice on its head. Repeat the process to ligate the other oviduct. After the surgery is complete, place the frog in shallow water, keeping its skin moist and its nose exposed to prevent drowning. Allow the animal to recover slowly from the anesthesia for temperature acclimation before transferring to a larger volume of water at room temperature. If the lung is accidentally damaged during the surgery, the frog will suffer gas exchange problems and show substantial gaseous edema. The frog can be used for egg production immediately after recovery from the anesthesia or returned to its tank to be used at a later time. The normal protocol for the induction of ovulation is used in obtaining coelomic eggs. Eggs can be recovered from the coelomic cavity of a surgically ligated animal 12 hours after HCG injection. The frog is anesthetized as before and laid ventral side up on a cheesecloth-covered tray of ice. Cut away the abdominal skin in the form of a three-sided flap (two ventral and one posterior cut) and drape the flap over the animal’s head. Make lateral cuts in the muscle
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FIG.8. The body cavity of a female showing the morphology of the internal organs and the oviduct. FIG.9. The pars recta-pars convoluta oviduct junction and the ostium of the oviduct where coelomic eggs tend to accumulate.
of the body wall on each side of the skinned area. Do not cut through the midline of the body wall muscle as the anterior abdominal vein will be severed and blood will contaminate the pleuroperitoneal cavity. With a spoon-shaped spatula, scoop the eggs from the body cavity and place them in DeBoers or Tris-DeBoers solution. Most of the eggs will be located in the anterior portion of the body cavity in the vicinity of the ostium (Figs. 8 and 9). The animal can be sacrificed by severing its spinal column or removing its heart. The recovered coelomic eggs are washed with DeBoers solution, lysed, and the CEs isolated by sieving. The CE is composed of six glycoproteins as indicated in Fig. 1.
G. Obtaining Oocytes for Ovarian Envelope Preparation To obtain oocytes, ovaries are excised from adult females 96 hours after injecting 35 IU of PMSG. The females are sacrificed by severing the spine. The ovaries are washed with Tris-DeBoers solution, pH 7.4, to remove blood; fat and connective tissue are dissected away. The tissue is then processed through an industrial meat grinder (5 mm diameter holes in the template) to rupture the follicles. The ground tissue, containing numerous individual oocytes, is sieved through a 10-mesh stainless steel screen by gently shaking the screen while washing the oocytes with Tris- DeBoers solution, pH 7.4. Connective tissue clumps of oocytes are retained by the screen. The sieved fraction containing individual oocytes is washed free of egg lysate by suspending the
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JERRY L. HEDRICK AND DANIEL M. HARDY
cells in buffer, sedimenting them at unit gravity, and decanting the supernatant solution. The washed oocytes are then crudely fractionated according to size by repeatedly resuspending the cells in buffer, allowing them to settle briefly, and decanting the larger, more buoyant cells. In this way, a fraction enriched in late stage oocytes is obtained. Although digestion of ovarian tissue with collagenase is a commonly used method of preparing Xenopus laevis oocytes, we found this enzymatic method unsuitable for preparing oocytes for O E isolation. Clostripain is a contaminant of collagenase preparations, and it proteolyzes gp43 of the O E (D. M. Hardy and J. L. Hedrick, unpublished observations). Thus, OEs obtained from collagenase-disrupted ovaries appear compositionally similar to the VE, an artifact which is avoided if the ovaries are disrupted mechanically. Inhibiting clostripain with thiol-reactive reagents such as iodoacetamide does not solve this problem as the clostripain activity appears to be necessary for effective digestion of the ovary into individual cells. Using the tissue grinding method, we isolated OEs from up to 106 frogs (1.93 kg of ovaries) with a yield of 40 ml of packed envelopes representing 140 mg of envelope protein.
H. Radioiodination of Isolated Envelopes Radiolabeling of the envelope glycoproteins with I is useful for various experiments which require high sensitivity detection methods such as the topological location of envelope glycoproteins, Western blotting, and spermenvelope binding experiments (Nishihara et al., 1983; Lindsay and Hedrick, 1988). The envelopes are readily labeled using IODO-GEN@(Pierce, Rockford, 11) and lZ5Iby the method of Markwell and Fox (1978). The envelopes can be iodinated in the form of intact particulate envelopes, heat-solubilized envelopes (large molecular weight supramolecular complexes of envelope glycoproteins), or as dissociated solubilized individual glycoproteins (SDS or guanidine-HC1 dissociated). Envelopes are solubilized by heating an envelope suspension to 70"- 80°C in water or an appropriate buffer adjusted to pH 8-9 for 10 minutes. After cooling, the solution is neutralized with dilute HCl or acetic acid. Dissociation of the envelopes is effected by heating an envelope suspension at 100°C for 90 seconds in a buffer solution containing 1-2% SDS or 6 M guanidineHCl at pH 7-8. The envelope suspension/solution (250 pg of protein) is transferred to a 10 x 75 mm culture tube plated with 50 pg of IODO-GEN@ (Pierce, Rockford, 11). Approximately 500 pCi of carrier-free '"I is added to initiate the reaction. After 15-20 minutes at room temperature, the free lZ5Iis separated from '251-derivatized glycoproteins by washing particulate
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envelopes with ice-cold water 5-6 times by centrifugation (1500 g for 5 minutes). For solubilized envelopes, the free '1 is removed by gel-filtration chromatography using BioGel P-2 or Sephadex G-25 (room temperature and an appropriate buffer, e.g., DeBoers or Tris-DeBoers, pH 7.8).
111. Discussion
A. Jelly Coat Layers The initial report on the number of Xenopus laeuis egg jelly coat layers stated the presence of three layers (Freeman, 1968). More recent light microscopic studies indicated the possible presence of a subdivision of jelly coat layer J3 into two separate layers in dilute DeBoers solution (Yoshizaki, 1985). This report awaits confirmation by other methods and has apparently not been pursued further. Fine structure studies on the jelly coat layers using contemporary methods remain to be done. The gross chemistry and glycoprotein composition of the jelly coat layers has been determined (Yurewicz et al., 1975). Analysis using gel electrophoretic, immunological, and ultracentrifugal methods have established that at least nine different glycoproteins are present in all three layers with J,, J2, and J3 having two or three, two, and four or five macromolecules, respectively (Table I). Isolation of individual glycoproteins has yet to be accomplished, which is a necessary preliminary for structure-function studies employing recombinant DNA methods and determining the chemical structures of functionally important oligosaccharides. In addition the location of individual glycoproteins within jelly coat layers (they are undoubtedly not uniformly distributed) using immunocytochemical or conjugated lectin methods has not been done. The specific roles of jelly coat glycoproteins in fertilization and early development is largely speculative although the importance of the jelly coat to fertilization was experimentally demonstrated more than 100 years ago (Newport, 1851). A specific hypothesis for the biological function of jelly coat glycoproteins was proposed for Bufo japonicus, namely, that the glycoproteins serve to bind Ca2+ and Mg2+,ions which are necessary for the sperm acrosome reaction (for discussion, see Katagiri, 1987). The generality of this hypothesis in other amphibians, and in particular Xenopus laeuis, lacks experimental support, however.
B. Envelopes Ultrastructural differences in the various forms of the egg envelope correlate with functional changes in the envelope in regard to sperm penetration.
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The organizational elements of the envelope are predominantly fibrous in nature (Hedrick and Nishihara, 1991; Larabell and Chandler, 1991). The O E is constructed of fasiculated fibers which are penetrated by the macrovillar processes of the cumulus cells and the microvillar processes of the oocyte. The villar processes are important for transfer of macromolecules into the oocyte, for example, vitellogenin. The ovulated egg envelope, the CE, lacks the villar processes but retains the fasiculated or bundled fibrous appearance and is impenetrable to sperm (Grey et al., 1977). The VE of the oviposited egg has a dispersed fibrous appearance, as though the fiber bundles were untied and scattered. The VE is penetrable by sperm. The most notable ultrastructural change in the FE of the fertilized egg is the appearance of the fertilization layer on the outer aspect of the envelope. The envelope is again rendered impenetrable to sperm by the VE to F E transformation. Recent transmission electron microscopy studies have identified several new structures in or associated with the envelopes and one structural reorganization event of envelope fibers. A cloudlike prefertilization layer was identified by Yoshizaki and Katagiri (1984)situated between the outer aspect of the VE and the inner aspect of jelly coat layer J1 (Fig. 2). The prefertilization layer was proposed as being a secretory product of the pars recta oviduct and to function as a precursor to the fertilization layer (Yoshizaki, 1984). Presumably, it is the ligand for the cortical granule lectin which, together with the lectin, forms the fertilization layer (Nishihara et al., 1986). However, this layer or the glycoprotein(s) of which it is composed (and found in mercaptansolubilized jelly; Wyrick et al., 1974) have not yet been isolated and demonstrated to be the ligand for the cortical granule lectin. Interestingly, the jelly coat layers of Bufo japonicus eggs are immunologically related to the jelly coat layers of Xenopus laevis and also contain a ligand for the Xenopus laevis cortical granule lectin (Hedrick and Katagiri, 1988). Using quick freeze, deep etch replica methods, Larabell and Chandler have identified a new structure in oviposited eggs (for review, see Larabell and Chandler, 1991). A horizontal filament layer located at the tips of the perivitelline space microvilli and in intimate contact with the inner aspect of the VE was observed in oviposited eggs. The function of this layer and its chemical/macromolecular nature has not yet been determined. It is not known if the layer is associated with isolated VEs. Upon fertilization, the horizontal filamentous layer is converted into a smooth layer, still located on the tips of the egg microvilli. Again, as with the horizontal filament layer, the function and macromolecular composition of the smooth layer are unknown. A rearrangement of the fibers composing the VE component of the FE, VE*, into concentric sheets interconnected by fine filaments occurs. The function of this fiber rearrangement is unknown, but since the VE* also is impenetrable to sperm (Grey et al., 1976),this structural rearrangement may relate to the block
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to polyspermy functions of the FE. Larabell and Chandler also described an intricate network of filaments within the perivitelline space which interconnects the microvilli with each other, the egg plasma membrane, and the VE. The presence of organized structures within the perivitelline space was hinted at, but could not be conclusively demonstrated by, thin section studies (Grey et al., 1974). The molecular mechanisms involved in envelope conversions have been defined. Presumably conversion of the OE to the CE, when the villar processes retract from the envelope, is a purely physical phenomenon since no macromolecular differences between the envelopes have been detected and since the three-dimensional netlike structure of the fibrous envelope is flexible and deformable (Bakos et al., 1990a). The CE to VE conversion takes place as the egg transits the oviduct. The molecular mechanisms involved are addition of a new glycoprotein, gp57 (of unknown cellular source and unknown function), limited proteolysis at the C terminal end of gp43 to gp41, and subsequent conformational changes in the envelope glycoproteins as shown by dye binding, solubility, chemical modification, and deformability studies (Fig. 1; Bakos et al., 1990a,b). The pars rectasecreted protease was isolated and shown to be a 66K serine active site protease with homology to the chymotrypsin family of proteases (Hardy and Hedrick, unpublished observations). The isolated protease will convert the CE to the VE as shown by SDS-polyacrylamide gel electrophoresis of the envelope glycoproteins, ultrastructural changes as determined by electron microscopy, and alteration of envelope solubility. The VE to FE conversion takes place at fertilization and involves factors derived from the cortical granules. The molecular mechanisms involved are limited proteolysis of the gp69 and gp64 envelope components at their Cterminal ends by serine active site proteases specific for Arg and/or Phe amino acid residues (Fig. 1; Lindsay and Hedrick, 1989). The chymotrypsin-like protease appears to be located in an inactive form associated with the fibers of the perivitelline space. It is activated by the presumably cortical granuleassociated trypsinlike activity released in the cortical reaction (L. Lindsay, C. Larabell, and J. Hedrick, unpublished observations). This limited proteolysis of gp69 and gp64 is accompanied by or causes a subsequent conforrnational change in the envelope components (perhaps the formation of concentric sheets in the VE* observed by Larabell and Chandler, 1991),which is experimentally demonstrated by the same methods used to detect conformational changes in the CE to VE transformation (Bakos et al., 1990a).The fertilization layer is formed by a lectin-ligand binding reaction between the cortical granule lectin and its ligand (Nishihara et al., 1986).The lectin-ligand complex presumably binds to an envelope glycoprotein which prevents the fertilization layer from being lost during isolation (Hedrick and Nishihara,
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1991). The VE* glycoprotein involved in adhering the fertilization layer to the envelope has not yet been identified. It is apparent that the functional properties of the egg ECM are related to the ECM structure from the level of the macromolecule to the supramolecular complex. Isolation and determination of the structures of the individual glycoproteins of the egg jelly coat layers and the egg envelopes and relation of their giycoprotein structures to their gamete or zygote functions are necessary processes in understanding the role of the extracellular matrix in Xenopus laevis fertilization and development. ACKNOWLEDGMENTS Previously unpublished work reported here was supported by U.S. Public Health Service. Research Grant HD04906 (J.L.H.) and a National Research Service Award, HD07088 (D.M.H.). We thank N. J. Wardrip and M. N. Oda for assistance with Fig. 1 and Fig. 2.
REFERENCES Bakos, M., Kurosky, A., and Hedrick, J. L. (1990a).Biochemistry 29,609-615. Bakos, M., Kurosky, A., and Hedrick, J. L. (1990b).Deu. Biol. 138, 169-176. Carroll, E. J., and Hedrick, J. L. (1974). Deu. B i d . 38, 1-13. Elinson, R. P. (1973). J. Exp. Zool. 183,291-302. Elinson, R. P. (1986). Int. Rev. Cytol. 101, 59-100. Freeman, S. B. (1968). Biol.Bull. 135, 501-513. Gerton, G. L., and Hedrick, J. L. (1986a).J. Cell. Biochem. 30,341-350. Gerton, G. L., and Hedrick, J. L. (1986b).Deu. B i d . 116, 1-7. Gerton, G. L., Wardrip, N. J., and Hedrick, J. L. (1982).Anal. Biochem. 126, 116-121. Grey, R. D., Wolf, D. P., and Hedrick, J. L. (1974). Deu. B i d . 36,44-61. Grey, R. D., Working, P.K., and Hedrick, J. L. (1976). Deu. Bid. 54, 52-60. Grey, R. D., Working, P. K., and Hedrick, J. L. (1977). J. Exp. 2001.201,73-84. Gusseck, D. J., and Hedrick, J. L. (1971).Deu. Biol. 25,337-347. Hedrick, J. L., and Katagiri, C. (1988).J . Exp. Zool. 245, 78-85. Hedrick, J. L., and Nishihara, T. (1991).J . Electron Microsc. Technique 17,319-335 Hedrick, J. L., Smith, A. J., Yurewicz, E. C., Oliphant, G., and Wolf, D. P. (1974).Biol. Reprod. 11, 534-542. Katagiri, C. (1961).J. Fac. Sci. Hokkaido Uniu. Ser. Vl, Zool. 14,607-613. Katagiri, C . (1974).J. Embryol. Exp. Morphol. 31, 573-581. Katagiri, C. (1987). Zool. Sci. 4, 1-14. Larabell, C. A., and Chandler, D. E. (1991).J. Electron Microsc. Technique 17,294-318. Lindsay, L. L., and Hedrick, J. L. (1988).J. Exp. Zool. 245, 286-293. Lindsay, L. L., and Hedrick, J. L. (1989).Deu. Biol. 135,202-211. Markwell, M. A. K., and Fox, C. F. (1978). Biochemistry 17,4807-4817. Monk, B., and Hedrick, J. L. (1986). Zool. Sci. 3,459-466. Newport, G. (1851).Philos. Trans. R. Soc. London (Part 1, First Series) 141, 169-242. Nishihara, T., Gerton, G. L., and Hedrick, J. L. (1983).J . Cell. Biochem. 22,235-244.
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Nishihara, T., Wyrick, R. E., Working, P. K., Chen, Y., and Hedrick, J. L. (1986). Biochemistry 25,6013-6020. Schmell, E. D., Gulyas, B. J., and Hedrick, J. L. (1983). In “Mechanism and Control of Animal Fertilization” (J. F. Hartmann, ed.), pp. 365-413. Academic Press, New York. Wolf, D. P., and Hedrick, J. L. (1971).Deu. B i d . 25, 348-359. Wolf, D. P., Nishihara, T., West, D. M., Wyrick, R. E., and Hedrick, J. L. (1976). Biochemistry 15,3671-3678. Wyrick, R.E., Nishihara,T., and Hedrick, J. L. (1974).Proc. Natl. Acad. Sci. U.S.A. 71,2067-2071. Yamaguchi, S., Hedrick, J. L., and Katagiri, C. (1989). Deu. Growth Difler. 31,85-94. Yoshizaki, N. (1984). Deu. Growth DSffer. 26, 191-195. Yoshizaki, N. (1985).J . Morphol. 184, 155-169. Yoshizaki, N., and Katagiri, C. (1984).Zoo/. Sci. I, 255-264. Yurewicz, E. C., Oliphant, G., and Hedrick, J. L. (1975). Biochemistry 14,3101-3107.
Isolation of Extracellular Matrix Structures from Xenopus laevis Oocytes, Eggs, and Embryos JERRY L. HEDRICK Department of Biochemistry and Biophysics University of California at Davis Davis, CaIifornia 35616
DANIEL M. HARDY Howard Hughes Medical Institute University of Texas Southwestem Medical Center Dallas, Texas 75235
I. Introduction 11. Methods
A. B. C. D. E.
Induction of Ovulation and Obtaining Oviposited Eggs Preparation of Egg Jelly Coat Layers Sieving Methods for Isolation of Egg Envelopes Obtaining Oviposited Eggs for Vitelline Envelope Preparation Obtaining Activated or Fertilized Eggs for Fertilization Envelope Preparation F. Obtaining Coelomic Eggs for Coelomic Envelope Preparation G. Obtaining Oocytes for Ovarian Envelope Preparation H. Radioiodination of Isolated Envelopes 111. Discussion A. Jelly Coat Layers B. Envelopes References
23 1 METHODS IN CELL BIOLOGY. VOL. 36
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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JERRY L. HEDRICK A N D DANIEL M. HARDY
I. Introduction The extracellular matrix (ECM) surrounding amphibian eggs is composed of jelly coat layers, the egg envelope, and the fibrous elements of the perivitelline space (Fig. 2). The biological functions of the egg ECM in fertilization and development were first noted more than a century ago when Newport (1851) reported that unjellied eggs recovered from the coelom were not fertilizable. We now appreciate that the oviductally produced jelly coat layers of amphibian eggs are required for the process of sperm capacitation and/or induction of the sperm acrosome reaction (for review, see Hedrick and Nishihara, 1991; Katagiri, 1987). The oviposited egg envelope provides an initially penetrable structure to the sperm which is subsequently modified by products released in the cortical reaction to a sperm-impenetrable structure in the block to polyspermy reaction (for reviews, see Schmell et al., 1983; Elinson, 1986). However, the ovulated egg recovered from the coelom possesses an envelope which is also impenetrable to sperm as originally shown in Rana pipiens and Bufo japonicus (Elinson, 1973; Katagiri, 1974). Thus, the egg envelope goes from a sperm-impenetrable state (the coelomic egg) to a penetrable state (the oviposited egg) and back to an impenetrable state (the fertilized egg or zygote). At fertilization the egg envelope is chemically modified and its macromolecular permeability properties changed (discussed in Schmell et al., 1983). This permeability change (hardening) results in an osmotically driven envelope elevation, owing to the influx of water into the perivitelline space. The jelly after fertilization functions as a “sticky substrate” for the adherence of the zygote to objects in its surroundings, protects the zygote against physical damage, and also provides a microbiological barrier (bacteria are rarely found within the jelly coat layers). Thus, after fertilization the ECM protects the developing embryo and functions as a barrier for the chemical and biological regulation of the embryo environment. This protective function of the ECM persists until the embryo develops into a free swimming tadpole and hatches from the ECM. The hatching process involves both physical and enzymatic mechanisms (Carroll and Hedrick, 1974).The ECM of the egg and the embryo, therefore, play significant roles in fertilization and development. Contemporary approaches to the structure-function relations of the ECM and its component macromolecules using Xenopus laeuis have utilized electron microscopic, biochemical, and immunological methods (for reviews, see Hedrick and Nishihara, 1991; Larabell and Chandler, 1991). The macromolecular compositions of the jelly coat layers (Table I) and the various forms of the egg envelopes have been determined (Fig. 1). Isolation of the individual glycoproteins composing the envelopes is currently in progress and
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TABLE I GLYCOPROTEIN COMPOSITION OF Xenopus laeuis JELLY COAT LAYERS'
Number of different glycoproteins Jelly coat layer Jl
J, J3
Total
Electrophoresis ID
SVC
IM
CA
SDS-A
2 2
2 2 4 8
3 2 4 9
2 2 5 9
3 2
4
8*
4 9'
a Abbreviations: ID, immunodiffusion; SVC, sedimentation velocity centrifugation; IM, immuno; CA, cellulose acetate; SDS-A, sodium dodecyl sulfate-agarose. Adapted from Yurewicz et al., 1915. One additional component common to J, and J3 was detected. 'Two additional components common to J , and J, were detected.
is a necessary prerequisite to the use of recombinant DNA methods for determination of the structure-function relations of the ECM glycoproteins (Gerton et al., 1982; Nishihara et al., 1986; Lindsay and Hedrick, 1988).
11. Methods
A. Induction of Ovulation and Obtaining Oviposited Eggs The quality and quantity of eggs and the timing of ovulation are greatly improved by using a pregnant mare serum gonadotropin (PMSG) priminghuman chorionic gonadotropin (HCG) ovulating protocol over that which uses HCG alone (Hedrick and Nishihara, 1991). Accordingly, females kept on a 12 hours light/l2 hours dark schedule at 20°-23"C are injected into the dorsal lymph sac with 35 IU of PMSG dissolved in 1 ml of 0.15 M NaCl. After 96 hours, ovulation is induced by the injection into the dorsal lymph sac of 500-1000 IU of HCG. After 5-6 hours, eggs are stripped from the females into DeBoers solution. DeBoers solution is 110 mM NaCl, 1.3 mM CaCl,, and 1.3 mM KCl, adjusted to pH 7.2-7.8 with NaHCO, (Katagiri, 1961).Normally, three or four strippings are done at 1.5- to 2-hour intervals and 1500-3000 eggs per female collected. Females are returned to their tanks and rested for at least 6 weeks before they are used again for egg production.
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B. Preparation of Egg Jelly Coat Layers 1.
COLLECTION OF INDIVIDUAL JELLYCOAT LAYERS
Individual jelly coat layers can be microdissected from DeBoers-washed eggs using a pair of sharp watchmaker’s forceps and a dissecting microscope (Yurewicz et al., 1975). A slight hydration of the jelly coat layers in DeBoers solution aids in their removal. This is usually accomplished by allowing the oviposited eggs to sit in DeBoers solution for 20-30 minutes at room temperature, The use of dilute salt solutions in this procedure is not possible as jelly coat layer J, becomes so sticky that manipulation of the eggs is difficult. However, for the recovery of eggs with only J, attached, this stickiness can be a benefit as the eggs adhere to the bottom of a culture dish and do not otherwise have to be held in place. The soft outermost layer, J3, can be cleanly removed from the tougher and more leatherlike J2 layer by gently stripping away J3 with one pair of forceps while using a second pair to hold the egg. Isolation of J2 requires piercing layer J2 with one pair of forceps and then teasing the torn J2layer away from the soft and highly hydrated J, layer with a second pair of forceps. J2 is invariably dissected with small amounts of adhering J, . Because of its soft, sticky “chewing gum” nature, J1 is most easily separated from the egg by solubilization in mercaptan solutions. Accordingly, J 3 - and J,-less eggs are immersed in 100 mM NaC1-50 mM Tris-HC1, pH 8.0, containing 2-5 mM dithiothreitol. The eggs are gently swirled, and dejellying is usually complete in 5-lo minutes at 22°C (as observed with the naked eye or with a dissecting microscope). The egg and vitelline envelope (VE) remain intact during this period. However, continued exposure to the dithiothreitol will dissolve the VE and lyse the egg. Decant the solution from the eggs and adjust the pH of the jelly solution to 6.5-7.0 for storage (oxidation of the sulfhydryl groups to disulfides occurs very slowly at neutral or acidic pH values; for long-term storage, 1 mM EDTA should also be added). A variety of mercaptans, such as mercaptoethanol, can be used instead of dithiothreitol, and DeBoers or other salt solutions can replace the NaC1-Tris solution. It should be noted that the rate of jelly dissolution is a function of the solution pH and mercaptan concentration (Gusseck and Hedrick, 1971). Lower pH values will slow the rate, and higher pH values will increase the rate of the dissolution reaction. The isolated insoluble jelly coat layers 5, and J2 can be washed in appropriate salt solutions and then dissolved in dithiothreitol solutions as was done with J, . The solubilized jelly coat layers are centrifuged at 12,000 g to remove particulate matter and dialyzed versus an appropriate buffer to adjust the pH and ion concentration. The jelly from a single egg contains
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235
approximately 41 pg of glycoprotein, with J , , and J , , and J, contributing 19, 5, and 17 pg, respectively. 2.
TOTALSOLUBILIZATION
OF
JELLY
For the preparation of jelly coat glycoproteins from all the jelly coat layers or for the preparation of dejellied eggs, oviposited eggs can be treated with mercaptan solutions. Place a monolayer of washed eggs in a flat-bottomed dish such as a crystallization dish (e.g., 20-30 ml of eggs in a 125 x 65 mm dish). Add 80 ml of 10 mM Tris-DeBoers solution adjusted to pH 8.9 and containing 45 mM mercaptoethanol and gently rock the dish. Dissolution of the jelly coat layers can be followed by the naked eye and should be complete in 3-5 minutes. As stated previously, continued exposure of the eggs to the mercaptoethanol solution will dissolve the VE and lyse the eggs. Decant the solubilized jelly and adjust the pH to 6.5-7.0. If the eggs are also to be recovered, rinse them several times in 10 mM Tris-DeBoers solution at pH 7.0-7.8. From 1000 eggs, approximately 41 mg of glycoprotein is recovered with a composition of 39% protein and 61 % carbohydrate. 3.
PREPARATION OF
35 S-LABELED
JELLY
The egg jelly contains sulfate esters with the majority, if not all, of the sulfate located in jelly coat layer J, (Yurewicz et al., 1975). [35S]Sulfate is readily incorporated into the J , jelly coat glycoproteins (Hedrick et al., 1974). Injection of 1-5 mCi into the dorsal lymph sac along with the HCG gives maximum incorporation into the egg ECM. Approximately 0.1% of the injected dose is incorporated with 3000-4500cpm/egg of which 42% is associated with the jelly and 58% with the egg. The jelly contains both free and glycoprotein-bound [35S]sulfate with the distribution ranging from 76 and 24%, respectively, from eggs collected in the first stripping to 33 and 67%, respectively, from eggs collected in the final stripping. The specific activity of thejelly is also a function of the stripping time, with the highest specific activity obtained in the last stripping. A second ovulation of the female 5-8 days later with an additional 2-5 mCi of [35S]sulfate will give smaller numbers of eggs but with higher specific activity of the jelly glycoprotein. Some 93% of the [35S]sulfate counts are incorporated into a single acidic glycoprotein in jelly coat layer J,.
C. Sieving Methods for Isolation of Egg Envelopes All forms of the egg envelope [VE, fertilization envelope (FE), coelomic envelope (CE), and ovarian envelope (OE)] are isolated using a common
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JERRY L. HEDRICK AND DANIEL M. HARDY
sieving method (Wolf et al., 1976).Jellyless eggs are poured into a large syringe (10-100 ml depending on the number of eggs used) and lysed by passage through an 18-gauge needle. The lysate is filtered through a 210-pm nylon screen suspended on the top of a wide-mouth bottle. Envelopes resting on the top of the screen are gently washed through the screen with a wash bottle containing ice-cold distilled water or 10 mM Tris-DeBoers solution, pH 7.0. This step removes the larger debris. The filtered envelope suspension is then poured through a 105-pm screen and the smaller particulate matter is washed through as with the above procedure. This sieving process is repeated (usually twice) until the envelopes are visually free from contaminating particulate material. The purified envelopes are washed from the screen into a conical centrifuge tube and collected by a 1- to 2-minute centrifugation in a clinical centrifuge (room temperature, 1500 9). The major envelope contaminant is yolk granules, which are solubilized by overnight storage of the envelopes at 4°C in a salt solution (2 M NaCl, 0.2 M imidazole-HC1,2mM CaCl, , pH 7.0; Lindsay and Hedrick, 1989). The particulate envelopes are collected by centrifugation and washed with ice-cold water, DeBoers solution, or 0.15 M NaCl, depending on the subsequent use of the envelopes. The envelopes are stored at 4"C, pH 7.0, in high salt solutions (0.1 M NaCl) or CaCl, solutions to prevent swelling of the envelopes and solubilization of envelope glycoproteins, particularly the gp57 component of the VE and the FE (Bakos et d., 1990a). Envelopes can be solubilized by heating for 10 minutes at 80°C in water adjusted to pH 9 with Tris or NaOH. The envelopes remain soluble when the solution pH is subsequently lowered to pH 7 with dilute HCl or acetic acid.
D. Obtaining Oviposited Eggs for Vitelline Envelope Preparation Oviposited eggs are collected using the hormonal stimulation methods stated above. The oviposited eggs are dejellied, lysed, and VEs collected by the sieving methods described above. Approximately 1 mg of glycoprotein (90% protein, 10% carbohydrate) is obtained per 1000 eggs. The VE is composed of seven glycoproteins as indicated in Fig. 1.
E. Obtaining Activated or Fertilized Eggs for Fertilization Envelope Preparation Jellied oviposited eggs are activated by adding the calcium ionophore A23187 (2-5 p M final concentration) in 0.05 DeBoers or 0.05 TrisDeBoers solution (Monk and Hedrick, 1986).The calcium ionophore A23187
12.
120
112
69
66
64
61
57
43
41
37
I
I
I
I
I
I
I
I
I
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I
I
I
I
I
I
I
I
I
r
r
r
Q
sho
6
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100
?8
237
ISOLATION OF EXTRACELLULAR MATRIX STRUCTURES
.3
$
1
0.1
t 0.01
FE
I
I
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120
112
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37
Envelope Glycoprotein FIG. 1. The glycoprotein composition of egg envelopes. The glycoproteins are noted on the abcissa without the prefix gp. For graphing reasons (log of the weight %), 0% is equivalent to 0.01%. (The glycoprotein composition of CE is from Gerton and Hedrick, 1986b;that of VE and FE is from Gerton and Hedrick, 1986a; quantification by image analysis is from Hedrick and Nishihara, 1991.)The glycoprotein composition of the OE is qualitatively the same as the CE (data not shown).
stock solution (2-5 mM) is prepared by dissolving the ionophore in absolute ethanol or dimethyl sulfoxide. The activated eggs are gently swirled at room temperature, and after 20 minutes the medium is decanted. The activated eggs are washed, dejellied, lysed, and the activated FEs collected by sieving. Approximately 1.3 mg of glycoprotein (88% protein, 12% carbohydrate) is obtained per 1000 eggs (Wolf et d.,1976).The FE is composed of at least nine glycoproteins as indicated in Fig. 1. An alternative method for preparing the FE is to fertilize eggs (Wolf and Hedrick, 1971).A single testis is macerated in a conical centrifuge tube with a glass rod in 1 ml of DeBoers solution (the sperm concentration is approximately 1 x lo7 cells/ml). DeBoers solution-washed eggs are spread in a monolayer in the bottom of a dish. The solution is decanted and replaced with 0.05 DeBoers or 0.05 Tris-DeBoers solution. Sperm are immediately
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JERRY L. HEDRICK AND DANIEL M. HARDY
added to a final concentration of at least 1 x 105/ml. The suspension is gently swirled, and, after 20 minutes at room temperature, the overlying solution is decanted, the eggs dejellied and lysed, and FEs collected by sieving. The fertilization layer can be removed from the FE by solubilization to obtain the activated egg envelope moiety, VE* (the vitelline envelope component of the FE). Isolated FEs are suspended in 0.5 M galactose, 1.0 mM CaCl,, and 10 mM Tris-HC1, pH 7.8, for 2 hours at room temperature (Nishihara et al., 1983). Alternatively, the fertilization layer can be solubilized with 5 mM EDTA, 10 mM Tris-HCI, 154 m M NaCI, pH 7.8 (Hedrick and Nishihara, 1991). The VE* is recovered by centrifugation (5000 g for 5 minutes), extracted an additional time with the galactose- or EDTAcontaining solution, and finally washed by centrifugation in ice-cold water or a buffer solution. The extract contains the solubilized fertilization layer components, namely, the cortical granule lectin and its ligand (Nishihara et al., 1986).
F. Obtaining Coelomic Eggs for Coelomic Envelope Preparation To obtain coelomic eggs, the oviducts are surgically ligated prior to the induction of ovulation (Bakos et al., 1990b). A female frog is anesthetized by immersion in 1 liter of MS222 (1.6 g of ethyl 3-aminobenzoate methane sulfonate/liter) at 0°C for 15-20 minutes. Arrange the frog on a cheeseclothcovered tray of ice (Fig. 3). By palpation, locate the posterior dorsal point of the scapula and make a 1-cm longitudinal incision through the skin about 1.5 cm posterior to the scapula and midway between the scapula and the lateral line (Fig. 3). The incision is best made by holding the skin taut with the fingertips and cutting with a scalpel. After cutting through the skin, the underlying muscle layers are lifted with forceps and cut with a pair of scissors to avoid cutting the underlying lung. There are three muscle layers, with the inner layer being very thin; cutting through it will expose the lung (Fig. 4). While holding the inner muscle layer with forceps, with a second pair of forceps, grasp the innermost muscle layer inside the incision in a dorsal direction, gently pull the muscle externally, and transfer the grasp of the second forcep to a pronged hemostat. This procedure will prevent the inner muscle layer from retracting into the incision. Gently pull the inner muscle layer through the incision (evert) little by little until the attachment of the oviduct to the muscle layer and the ostium of the oviduct is located. The pars recta oviduct is easily differentiated from the pars convoluta oviduct as the pars recta is heavily vascularized relative to the white, opaque pars convoluta (Fig. 5).
12.
ISOLATION OF EXTRACELLULAR MATRIX STRUCTURES
239
FIG.2. Diagram of the extracellular matrix of the oviposited Xenopus laeuis egg. The ECM structures in the diagram are not to scale. CG, Cortical granules; PS, perivitelline space; VE, vitelline envelope; PL, prefertilization layer; J,, J,, J 3 , individual jelly coat layers. Fia. 3. Location of the incision site for oviduct ligation.
FIG.4. Exposure of the pleuroperitoneal cavity. The three muscle layers have been cut and parted to expose the underlying lung. Fic. 5. The pars recta and pars convoluta oviduct. The pars recta is heavily vascularized compared to the pars convoluta (forceps at left). The pars recta is attached to the body wall (forceps at top) and leads to the ostium.
240
JERRY L. HEDRICK AND DANIEL M. HARDY
FIG.6. Ligating the oviduct. FIG.7. The sutured incision at the completion of the ligation procedure.
Use 5-0 silk suture or single-use hemostatic silver clips to ligate the oviduct (Fig. 6). It is important to ligate the oviduct as close to the ostium as possible. Release the pronged hemostat and tuck the ligated oviduct back into the body cavity by lifting the body wall with forceps. Suture the three muscle layers of the body wall with 4-0 gut or close the incision with surgical staples. The three layers do not have to be separately sutured, but they must be well sutured to prevent leakage of ovulated eggs through the incision. Suture the skin with 3-0 silk suture (Fig. 7). If the frog begins to recover prematurely from the anesthesia, place some flakes of ice on its head. Repeat the process to ligate the other oviduct. After the surgery is complete, place the frog in shallow water, keeping its skin moist and its nose exposed to prevent drowning. Allow the animal to recover slowly from the anesthesia for temperature acclimation before transferring to a larger volume of water at room temperature. If the lung is accidentally damaged during the surgery, the frog will suffer gas exchange problems and show substantial gaseous edema. The frog can be used for egg production immediately after recovery from the anesthesia or returned to its tank to be used at a later time. The normal protocol for the induction of ovulation is used in obtaining coelomic eggs. Eggs can be recovered from the coelomic cavity of a surgically ligated animal 12 hours after HCG injection. The frog is anesthetized as before and laid ventral side up on a cheesecloth-covered tray of ice. Cut away the abdominal skin in the form of a three-sided flap (two ventral and one posterior cut) and drape the flap over the animal’s head. Make lateral cuts in the muscle
12.
ISOLATION OF EXTRACELLULAR MATRIX STRUCTURES
24 1
FIG.8. The body cavity of a female showing the morphology of the internal organs and the oviduct. FIG.9. The pars recta-pars convoluta oviduct junction and the ostium of the oviduct where coelomic eggs tend to accumulate.
of the body wall on each side of the skinned area. Do not cut through the midline of the body wall muscle as the anterior abdominal vein will be severed and blood will contaminate the pleuroperitoneal cavity. With a spoon-shaped spatula, scoop the eggs from the body cavity and place them in DeBoers or Tris-DeBoers solution. Most of the eggs will be located in the anterior portion of the body cavity in the vicinity of the ostium (Figs. 8 and 9). The animal can be sacrificed by severing its spinal column or removing its heart. The recovered coelomic eggs are washed with DeBoers solution, lysed, and the CEs isolated by sieving. The CE is composed of six glycoproteins as indicated in Fig. 1.
G. Obtaining Oocytes for Ovarian Envelope Preparation To obtain oocytes, ovaries are excised from adult females 96 hours after injecting 35 IU of PMSG. The females are sacrificed by severing the spine. The ovaries are washed with Tris-DeBoers solution, pH 7.4, to remove blood; fat and connective tissue are dissected away. The tissue is then processed through an industrial meat grinder (5 mm diameter holes in the template) to rupture the follicles. The ground tissue, containing numerous individual oocytes, is sieved through a 10-mesh stainless steel screen by gently shaking the screen while washing the oocytes with Tris- DeBoers solution, pH 7.4. Connective tissue clumps of oocytes are retained by the screen. The sieved fraction containing individual oocytes is washed free of egg lysate by suspending the
242
JERRY L. HEDRICK AND DANIEL M. HARDY
cells in buffer, sedimenting them at unit gravity, and decanting the supernatant solution. The washed oocytes are then crudely fractionated according to size by repeatedly resuspending the cells in buffer, allowing them to settle briefly, and decanting the larger, more buoyant cells. In this way, a fraction enriched in late stage oocytes is obtained. Although digestion of ovarian tissue with collagenase is a commonly used method of preparing Xenopus laevis oocytes, we found this enzymatic method unsuitable for preparing oocytes for O E isolation. Clostripain is a contaminant of collagenase preparations, and it proteolyzes gp43 of the O E (D. M. Hardy and J. L. Hedrick, unpublished observations). Thus, OEs obtained from collagenase-disrupted ovaries appear compositionally similar to the VE, an artifact which is avoided if the ovaries are disrupted mechanically. Inhibiting clostripain with thiol-reactive reagents such as iodoacetamide does not solve this problem as the clostripain activity appears to be necessary for effective digestion of the ovary into individual cells. Using the tissue grinding method, we isolated OEs from up to 106 frogs (1.93 kg of ovaries) with a yield of 40 ml of packed envelopes representing 140 mg of envelope protein.
H. Radioiodination of Isolated Envelopes Radiolabeling of the envelope glycoproteins with I is useful for various experiments which require high sensitivity detection methods such as the topological location of envelope glycoproteins, Western blotting, and spermenvelope binding experiments (Nishihara et al., 1983; Lindsay and Hedrick, 1988). The envelopes are readily labeled using IODO-GEN@(Pierce, Rockford, 11) and lZ5Iby the method of Markwell and Fox (1978). The envelopes can be iodinated in the form of intact particulate envelopes, heat-solubilized envelopes (large molecular weight supramolecular complexes of envelope glycoproteins), or as dissociated solubilized individual glycoproteins (SDS or guanidine-HC1 dissociated). Envelopes are solubilized by heating an envelope suspension to 70"- 80°C in water or an appropriate buffer adjusted to pH 8-9 for 10 minutes. After cooling, the solution is neutralized with dilute HCl or acetic acid. Dissociation of the envelopes is effected by heating an envelope suspension at 100°C for 90 seconds in a buffer solution containing 1-2% SDS or 6 M guanidineHCl at pH 7-8. The envelope suspension/solution (250 pg of protein) is transferred to a 10 x 75 mm culture tube plated with 50 pg of IODO-GEN@ (Pierce, Rockford, 11). Approximately 500 pCi of carrier-free '"I is added to initiate the reaction. After 15-20 minutes at room temperature, the free lZ5Iis separated from '251-derivatized glycoproteins by washing particulate
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envelopes with ice-cold water 5-6 times by centrifugation (1500 g for 5 minutes). For solubilized envelopes, the free '1 is removed by gel-filtration chromatography using BioGel P-2 or Sephadex G-25 (room temperature and an appropriate buffer, e.g., DeBoers or Tris-DeBoers, pH 7.8).
111. Discussion
A. Jelly Coat Layers The initial report on the number of Xenopus laeuis egg jelly coat layers stated the presence of three layers (Freeman, 1968). More recent light microscopic studies indicated the possible presence of a subdivision of jelly coat layer J3 into two separate layers in dilute DeBoers solution (Yoshizaki, 1985). This report awaits confirmation by other methods and has apparently not been pursued further. Fine structure studies on the jelly coat layers using contemporary methods remain to be done. The gross chemistry and glycoprotein composition of the jelly coat layers has been determined (Yurewicz et al., 1975). Analysis using gel electrophoretic, immunological, and ultracentrifugal methods have established that at least nine different glycoproteins are present in all three layers with J,, J2, and J3 having two or three, two, and four or five macromolecules, respectively (Table I). Isolation of individual glycoproteins has yet to be accomplished, which is a necessary preliminary for structure-function studies employing recombinant DNA methods and determining the chemical structures of functionally important oligosaccharides. In addition the location of individual glycoproteins within jelly coat layers (they are undoubtedly not uniformly distributed) using immunocytochemical or conjugated lectin methods has not been done. The specific roles of jelly coat glycoproteins in fertilization and early development is largely speculative although the importance of the jelly coat to fertilization was experimentally demonstrated more than 100 years ago (Newport, 1851). A specific hypothesis for the biological function of jelly coat glycoproteins was proposed for Bufo japonicus, namely, that the glycoproteins serve to bind Ca2+ and Mg2+,ions which are necessary for the sperm acrosome reaction (for discussion, see Katagiri, 1987). The generality of this hypothesis in other amphibians, and in particular Xenopus laeuis, lacks experimental support, however.
B. Envelopes Ultrastructural differences in the various forms of the egg envelope correlate with functional changes in the envelope in regard to sperm penetration.
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The organizational elements of the envelope are predominantly fibrous in nature (Hedrick and Nishihara, 1991; Larabell and Chandler, 1991). The O E is constructed of fasiculated fibers which are penetrated by the macrovillar processes of the cumulus cells and the microvillar processes of the oocyte. The villar processes are important for transfer of macromolecules into the oocyte, for example, vitellogenin. The ovulated egg envelope, the CE, lacks the villar processes but retains the fasiculated or bundled fibrous appearance and is impenetrable to sperm (Grey et al., 1977). The VE of the oviposited egg has a dispersed fibrous appearance, as though the fiber bundles were untied and scattered. The VE is penetrable by sperm. The most notable ultrastructural change in the FE of the fertilized egg is the appearance of the fertilization layer on the outer aspect of the envelope. The envelope is again rendered impenetrable to sperm by the VE to F E transformation. Recent transmission electron microscopy studies have identified several new structures in or associated with the envelopes and one structural reorganization event of envelope fibers. A cloudlike prefertilization layer was identified by Yoshizaki and Katagiri (1984)situated between the outer aspect of the VE and the inner aspect of jelly coat layer J1 (Fig. 2). The prefertilization layer was proposed as being a secretory product of the pars recta oviduct and to function as a precursor to the fertilization layer (Yoshizaki, 1984). Presumably, it is the ligand for the cortical granule lectin which, together with the lectin, forms the fertilization layer (Nishihara et al., 1986). However, this layer or the glycoprotein(s) of which it is composed (and found in mercaptansolubilized jelly; Wyrick et al., 1974) have not yet been isolated and demonstrated to be the ligand for the cortical granule lectin. Interestingly, the jelly coat layers of Bufo japonicus eggs are immunologically related to the jelly coat layers of Xenopus laevis and also contain a ligand for the Xenopus laevis cortical granule lectin (Hedrick and Katagiri, 1988). Using quick freeze, deep etch replica methods, Larabell and Chandler have identified a new structure in oviposited eggs (for review, see Larabell and Chandler, 1991). A horizontal filament layer located at the tips of the perivitelline space microvilli and in intimate contact with the inner aspect of the VE was observed in oviposited eggs. The function of this layer and its chemical/macromolecular nature has not yet been determined. It is not known if the layer is associated with isolated VEs. Upon fertilization, the horizontal filamentous layer is converted into a smooth layer, still located on the tips of the egg microvilli. Again, as with the horizontal filament layer, the function and macromolecular composition of the smooth layer are unknown. A rearrangement of the fibers composing the VE component of the FE, VE*, into concentric sheets interconnected by fine filaments occurs. The function of this fiber rearrangement is unknown, but since the VE* also is impenetrable to sperm (Grey et al., 1976),this structural rearrangement may relate to the block
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to polyspermy functions of the FE. Larabell and Chandler also described an intricate network of filaments within the perivitelline space which interconnects the microvilli with each other, the egg plasma membrane, and the VE. The presence of organized structures within the perivitelline space was hinted at, but could not be conclusively demonstrated by, thin section studies (Grey et al., 1974). The molecular mechanisms involved in envelope conversions have been defined. Presumably conversion of the OE to the CE, when the villar processes retract from the envelope, is a purely physical phenomenon since no macromolecular differences between the envelopes have been detected and since the three-dimensional netlike structure of the fibrous envelope is flexible and deformable (Bakos et al., 1990a). The CE to VE conversion takes place as the egg transits the oviduct. The molecular mechanisms involved are addition of a new glycoprotein, gp57 (of unknown cellular source and unknown function), limited proteolysis at the C terminal end of gp43 to gp41, and subsequent conformational changes in the envelope glycoproteins as shown by dye binding, solubility, chemical modification, and deformability studies (Fig. 1; Bakos et al., 1990a,b). The pars rectasecreted protease was isolated and shown to be a 66K serine active site protease with homology to the chymotrypsin family of proteases (Hardy and Hedrick, unpublished observations). The isolated protease will convert the CE to the VE as shown by SDS-polyacrylamide gel electrophoresis of the envelope glycoproteins, ultrastructural changes as determined by electron microscopy, and alteration of envelope solubility. The VE to FE conversion takes place at fertilization and involves factors derived from the cortical granules. The molecular mechanisms involved are limited proteolysis of the gp69 and gp64 envelope components at their Cterminal ends by serine active site proteases specific for Arg and/or Phe amino acid residues (Fig. 1; Lindsay and Hedrick, 1989). The chymotrypsin-like protease appears to be located in an inactive form associated with the fibers of the perivitelline space. It is activated by the presumably cortical granuleassociated trypsinlike activity released in the cortical reaction (L. Lindsay, C. Larabell, and J. Hedrick, unpublished observations). This limited proteolysis of gp69 and gp64 is accompanied by or causes a subsequent conforrnational change in the envelope components (perhaps the formation of concentric sheets in the VE* observed by Larabell and Chandler, 1991),which is experimentally demonstrated by the same methods used to detect conformational changes in the CE to VE transformation (Bakos et al., 1990a).The fertilization layer is formed by a lectin-ligand binding reaction between the cortical granule lectin and its ligand (Nishihara et al., 1986).The lectin-ligand complex presumably binds to an envelope glycoprotein which prevents the fertilization layer from being lost during isolation (Hedrick and Nishihara,
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1991). The VE* glycoprotein involved in adhering the fertilization layer to the envelope has not yet been identified. It is apparent that the functional properties of the egg ECM are related to the ECM structure from the level of the macromolecule to the supramolecular complex. Isolation and determination of the structures of the individual glycoproteins of the egg jelly coat layers and the egg envelopes and relation of their giycoprotein structures to their gamete or zygote functions are necessary processes in understanding the role of the extracellular matrix in Xenopus laevis fertilization and development. ACKNOWLEDGMENTS Previously unpublished work reported here was supported by U.S. Public Health Service. Research Grant HD04906 (J.L.H.) and a National Research Service Award, HD07088 (D.M.H.). We thank N. J. Wardrip and M. N. Oda for assistance with Fig. 1 and Fig. 2.
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