Isolation of Organelles and Components from Sea Urchin Eggs and Embryos

CHAPTER 20

Isolation of Organelles and Components from Sea Urchin Eggs and Embryos Gary M. Wessel* and Victor D. Vacquier{ *Department of Molecular and Cell Biology and Biochemistry Brown University Providence, Rhode Island 02912 {

Center for Marine Biotechnology and Biomedicine Scripps Institute of Oceanography University of California San Diego La Jolla, California 92093

I. Overview II. Egg Jelly Molecules AVecting Sperm A. Introduction B. Purification of FSP and SG from Sea Urchin Egg Jelly: Preparing Crude Egg Jelly C.
-Elimination D. Pronase Digestion E. DEAE Cellulose Chromatography III. Isolation of the Vitelline Layer from Sea Urchin Eggs A. Introduction B. Isolation of Vitelline Layers by Homogenization C. Isolation of Thin Vitelline Layers IV. Isolation of the Cell Surface Complex and the Plasma Membrane–Vitelline Layer (PMVL) Complex from Sea Urchin Eggs A. Introduction B. Isolation of the Cell Surface Complex C. Separation of the Plasma Membrane–Vitelline Layer Complex from the Cortical Granules V. The Cytolytic Isolation of the Egg Cortex A. Introduction B. Cytolytic Isolation of the Egg Cortex VI. Isolation of Cortical Granules A. Introduction B. Preparation of Cortical Granule Lawns METHODS IN CELL BIOLOGY, VOL. 74 Copyright 2004, Elsevier Inc. All rights reserved. 0091-679X/04 $35.00

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VII. VIII. IX. X. XI. XII.

XIII. XIV.

XV. XVI.

Isolation of Yolk Platelets Isolation of Mitochondria from Eggs and Embryos Isolation of Plasma Membranes and Lipid Rafts from Eggs and Zygotes Isolation of Microsomes Containing the Endoplasmic Reticulum Nuclear Isolation Procedures Removal and Isolation of the Fertilization Envelope A. Removal of the Envelope during Embryogenesis B. Isolation of Fertilization Envelopes C. Prevent Fertilization Envelopes from Forming Isolation of Cilia from Embryos Isolation of Extracellular Matrices from Embryos and Larvae A. Isolation of Hyalin B. The Apical Lamina C. The Basal Lamina/Blastocoel Matrix Isolation of Sea Urchin Larval Skeletons Resources for the Isolation of Additional Organelles A. Mitotic Spindles References

I. Overview Although sea urchin eggs and embryos are only about 100 microns in diameter and about 50 ng in total mass, the numbers obtainable from each adult reach many millions. The eggs are each at an identical stage when shed, and embryos are remarkably synchronous during development. Thus, one can isolate significant amounts of organelles and components from a uniform population of cells for specific biochemical and molecular analysis. Because of the ability to isolate various embryonic cell-types during development, and the conservation of many of the specialized egg and embryo organelles, this embryo has served as a rich source for experimentation, and development of techniques for organelle isolation. This chapter documents many of the prevalent techniques in organelle and component isolation from sea urchin eggs and embryos. Isolation and manipulation of sperm is dealt with in a separate chapter (Chapter 21). As might be expected for this classic system, the techniques have evolved over the years and so, where possible, we give the current, general isolation scheme followed by reference to significant variations on this theme. We emphasize here the protocols that have been developed since the first volume of this Methods book on echinoderms (Schroeder, 1986).

II. Egg Jelly Molecules AVecting Sperm A. Introduction Sea urchin eggs of various species are from 80 to 120 m in diameter and are surrounded by a jelly coat that, when hydrated, can be roughly 0.5 egg diameters in thickness. The egg jelly coat has two functions: to protect the egg surface and to

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induce the sperm acrosome reaction. Quick-Freeze, deep-etch rotary shadowing electron microscopy has been applied to the egg jelly layer and to isolated egg jelly molecules. The egg jelly layer in situ is a dense meshwork of three-dimensional fibers. Isolated jelly macromolecules resemble fibrous pearls on a necklace (Bonnell and Chandler, 1990; Bonnell et al., 1994). Three types of molecules have been isolated from egg jelly that have profound eVects on sperm physiology: (1) sperm-activating peptides (Darszon et al., 2001; Garbers, 1989; Kaupp et al., 2003; Suzuki, 1995); (2) the fucose sulfate polymer (FSP; Hirohashi and Vacquier, 2002a; SeGall and Lennarz, 1979; Vacquier and Moy, 1997; Vilela-Silva et al., 2002); and (3) a sialoglycan that is a form of polysialic acid (Hirohashi and Vacquier, 2002b; Kitazume et al., 1994). Considerable research has also been accomplished on the jelly layer of starfish eggs, the molecules of which also have profound eVects on sperm (Hoshi et al., 2000).

1. Egg Jelly Peptides The sequences of approximately 100 egg jelly peptides from various echinoid species have been determined (Suzuki, 1995). The egg jelly peptide speract from sea urchins of the genera Strongylocentrotus, Hemicentrotus, and Lytechinus has the sequence GFDLNGGGVG. Resact, the egg jelly peptide from sea urchins of the genus Arbacia, has the sequence CVTGAPGCVGGGRLNH2. Nowadays, these peptides can be synthesized commercially at reasonable prices. Approximately 150 to 200 million years separate the genera Strongylocentrotus and Arbacia (Gonzales and Lessios, 1999), both speract and resact bind to sperm receptors to up-regulate ion channels that activate sperm respiration and motility (reviewed in Darszon et al., 2001). Speract can also act synergistically with FSP in the induction of the sperm acrosome reaction (Hirohashi and Vacquier, 2002c; Yamaguchi et al., 1989). Speract binds to a 77 kDa receptor protein in the sperm tail plasma membrane that, in turn, up-regulates guanylyl cyclase (Garbers, 1989). However, resact binds directly to the Arbacia sperm tail guanylyl cyclase to upregulate the enzyme. The concentration of cGMP increases and acts directly on cation channels that regulate chemo-attraction toward the egg (Garbers, 1989; Ward et al., 1985). A single Arbacia sperm cell can change its swimming behavior upon binding one molecule of resact (Kaupp et al., 2003). The Arbacia guanylyl cyclase was the first receptor guanylyl cyclase to be cloned (Tamura et al., 2001).

2. The Sialoglycan (SG) The SG can be isolated from egg jelly by DEAE chromatography after pronase digestion, or
-elimination of total egg jelly. If pronase digestion is used, at least one amino acid will remain on the SG chain. This residue can be utilized for the coupling of the SG to a solid support, such as beaded AYgel (BioRad). In Hemicentrotus pulcherrimus, the polysialic acid chains are O-glycosidically attached to a 180 kDa glycoprotein, whereas in S. purpuratus the size of the intact molecule is 250 kDa. The structure of the polysialic acid was determined to

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be (5-O-glycolyl-Neu5Gc2)n, which is usually abbreviated ‘‘Neu5Gc.’’ It has a degree of polymerization of 4 to 40 residues in H. pulcherrimus and 25 residues in S. purpuratus (Kitazume et al., 1994). Purified SG greatly potentiates the FSP-induced acrosome reaction, but by itself is totally noninductive. SG elevates intracellular pH without increasing intracellular calcium (Hirohashi and Vacquier, 2002b). SG must bind a yet unidentified receptor on the sea urchin sperm plasma membrane. The potentiation by SG of the FSP-induced acrosome reaction is the first physiological response to be found for a polysialic acid (Hirohashi and Vacquier, 2002b).

3. The Fucose Sulfate Polymer (FSP) Purified FSP of egg jelly can by itself induce the sperm acrosome reaction. FSP is known to bind REJ1 (receptor for egg jelly-1), a glycoprotein on the sperm plasma membrane localized to the acrosome and the flagellum (Vacquier and Moy, 1997). FSP opens two pharmacologically diVerent sperm calcium channels that are required for acrosome reaction induction (reviewed in Darszon et al., 2001). REJ1 contains the
1000 residue ‘‘REJ module’’ found in members of the polycystin-1 family of orphan receptors that are mutated in autosomal dominant polycystic kidney disease (Moy et al., 1996). FSPs from diVerent sea urchin species are species-specific inducers of the sperm acrosome reaction. Most FSPs are (3--L-fucopyranosyl-1)n homopolymers that diVer from each other in the position of sulfation at the 2-O- and 4-O-positions of the fucosyl unit. Females of S. purpuratus have one of two isotypes of FSP. One isotype is a trisaccharide repeat in which the first fucosyl residue is sulfated on the 2-O- and 4-O-positions and the second and third fucosyl residues are sulfated on only the 4-O-position. The second isotype of FSP of this species is a homopolymer of-1!3-linked fucosyl residues sulfated on the 2-O- and 4-O-positions (Alves et al., 1998). The species S. franciscanus has the simplest FSP, which consists of -L-1!3-fucopyranosyl residues sulfated at only the 2-O-position (Vilela-Silva et al., 1999). Females of S. droebachiensis also synthesize two diVerent, female specific FSPs, but in this species, the glycosidic bond linking fucosyl residues is -1!4-, which is highly unusual considering the evolutionary closeness of this species to S. purpuratus (Vilela-Silva et al., 2002). FSP is the only known pure polysaccharide that induces a signal transduction event in an animal sperm. The surprising finding is that the specific molecular recognition between sperm receptors and FSP is based on the pattern of FSP’s sulfation and the glycosidic linkage. B. Purification of FSP and SG from Sea Urchin Egg Jelly: Preparing Crude Egg Jelly 1. Female sea urchins are injected with 0.5 M KCl and spawned into Milliporefiltered seawater (MFSW) and the eggs with hydrated jelly coats are allowed to settle. The supernatant is then aspirated away and the settled egg mass is resuspended in an equal volume of MFSW. 2. A combination pH electrode is placed in the beaker and the egg suspension stirred rapidly by hand with a spatula while 0.1 N HCl is added dropwise. When

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the pH reaches 5.0, the stirring continues for 2 min before the dropwise addition of 0.1 N NaOH to carefully bring the pH back to between 7 and 8. 3. The egg suspension is poured into 50 ml conical plastic centrifuge tubes that are centrifuged in a hand-driven centrifuge at
900 rpm for 2 min. 4. The supernatant is carefully removed with a 10 ml pipette and centrifuged at 30,000g for 30 min (4  C). The supernatant is called ‘‘crude egg jelly’’ and is aliquoted and frozen at 20  C. It retains its acrosome reaction-inducing ability for years. The macromolecules of crude egg jelly do not stain with Coomassie blue. However, silverstained gels of crude egg jelly show over 10 bands, varying from 350 to 20 kDa (Vacquier and Moy, 1997). Future work should be directed toward the purification of these macromolecules and the study of their eVects on sperm physiology.

C. b-Elimination 1. Crude egg jelly is precipitated by the addition of an equal volume of 95% ethanol. The precipitate is collected by a 1000g centrifugation (5 min) and dissolved in distilled water.
-elimination is performed by addition of 0.05 N NaOH containing 1 M NaBH4 (final concentrations) and incubation at 45  C for 24 h. 2. An equal volume of 50 mM sodium acetate, pH 5.0, is then added and the final pH brought to 5.0 by the dropwise addition of 2 M acetic acid. Silver staining of SDS-PAGE shows that all protein is degraded by this treatment (Hirohashi and Vacquier, 2002a).

D. Pronase Digestion Crude egg jelly is dissolved in 50 mM Tris, 10 mM sodium azide, pH 7.5, and pronase or proteinase-K added to 0.05 mg/ml. Digestion is at 37  C for 6 to 18 h. The egg jelly polysacchairde chains are then recovered by precipitation after the addition of two volumes of 95% ethanol. E. DEAE Cellulose Chromatography 1. SG and FSP are then separated and purified from each other by DEAE cellulose chromatography. We prefer to use Whatman DE-52 Cellulose. 2. Either
-eliminated or pronase digested egg jelly polysaccharides are dissolved in 50 mM sodium acetate, pH 5.0, and loaded onto DEAE-cellulose equilibrated in this buVer. After washing with 10 column volumes of this buVer, a linear gradient of 0 to 3 M NaCl in 50 mM acetate, pH 5.0, is applied. For a 50 ml column of DEAE, a 200 ml gradient, collected as forty 5 ml fractions, will work well. 3. Fractions are tested for sulfated glycans in the metachromatic assay (Farndale et al., 1986), for neutral sugars by the phenol sulfuric acid assay (Dubois et al., 1956), and sialic acid by the thiobarbituric acid assay (AminoV, 1961). Two

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peaks of eluted material are obtained. The fractions eluting before 1.0 M NaCl contain the SG, and the fractions eluting between 2 and 3 M NaCl contain FSP. 4. Peak fractions of SG and FSP are pooled and precipitated by addition of an equal volume of 95% ethanol and chilling for more than 30 min. The precipitates are collected by low-speed centrifugation, dissolved in distilled water, dialyzed against distilled water, and stored at 20  C. As little as 1 g per ml of FSP will induce the acrosome reaction in approximately 50% of S. purpuratus sperm. The relative molecular mass of SG determined by gel filtration chromatography is approximately 100,000, and of FSP, approximately 1 million Daltons (Hirohashi and Vacquier, 2002a,b). The SG has a molar ratio of fucose to sialic acid of approximately 1:6 (Hirohashi and Vacquier, 2002b), whereas the FSP is essentially 100% fucose sulfate (reviewed in Vilela-Silva et al., 2002).

III. Isolation of the Vitelline Layer from Sea Urchin Eggs A. Introduction Transmission electron microscopy of the vitelline layer (VL) of unfertilized eggs shows this glycocalyx to be a lacy, fibrous extracellular matrix intimately bonded to the plasma membrane (Chandler and Heuser, 1980; Kidd, 1978; Kidd and Mazia, 1980). The VL cannot be separated from the unfertilized egg without destroying the egg. The VL appears to be attached to the plasma membrane by regularly spaced posts that embed into the plasma membrane. During normal fertilization, the exocytosing cortical granule protease (Haley and Wessel, 1999) cleaves the bonds between the VL and the plasma membrane, thus allowing the VL to elevate and act as a template for the formation of the fertilization envelopes (see Weidman and Kay, 1986, for a comprehensive review). Eight monoclonal antibodies, that reacted species-specifically with the outer surface of the VL, inhibited sperm binding in proportion to the amount of antibody bound (Gache et al., 1983). Other work showed that the VL contains over 20 macromolecules of between 30 and 370 kDa (Correa and Carroll, 1997; Niman et al., 1984). A proteomics approach should now be applied to identify all the proteins comprising the VL. B. Isolation of Vitelline Layers by Homogenization 1. The method described here is taken from Glabe and Vacquier (1977). Eggs of S. purpuratus are obtained by 0.5 M KCl injection and egg jelly coats dissolved by a 2 min exposure to pH 5.0 seawater, as previously described. All procedures are at 4  C. 2. The settled eggs are washed three times in excess volumes of fresh MFSW. At this step, the eggs may be treated, or not treated, for 5 min with 0.2 mM N-bromosuccinimide in seawater (NBS, pH 7.5) and then washed with excess

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MFSW. The mild oxidation provided by the NBS results in VL that isolate as intact envelopes which still retain their sperm-binding capacity and ultrastructural appearance (Glabe and Vacquier, 1977). If NBS is excluded, the VLs fragment during homogenization but their ultrastructure is still preserved. 3. After NBS treatment, the egg pellet is resuspended in 10 volumes of 20 mM EDTA, 50 mM sodium acetate (pH 6.0), 0.4% Triton X-100, 10 mM benzamidine, 0.1 mg/ml soybean trypsin inhibitor (SBTI), and 1 mM PMSF (phenylmethylsulfonylfluoride, made as a 1 M stock in acetone). 4. The egg suspension is immediately poured into a loose-fitting glass ball Thomas tissue grinder and several cycles of homogenization done with periodic microscopic observation; 100% egg lysis and VL liberation can be obtained. 5. The homogenate is centrifuged 800g for 10 min, the supernatant discarded, and the VL pellet washed in fresh homogenization medium. Egg nuclei are found at the bottom of the tube. The VLs are stable to high and low salt buVers at neutral pH. 6. Forty ml of dejellied S. purpuratus eggs yields about 1 ml of 800g packed VLs, which are about 50 to 60 mg protein (Glabe and Vacquier, 1977). SDSPAGE analysis shows many protein components from 25 to 250 kDa. VLs are 90 to 95% protein and 4% carbohydrate by weight. Because Triton X-100 is used in their isolation, they are devoid of lipid. Electron microscopy shows VLs have an average thickness of 30 nm and that the spacing of the microvillar casts of the egg surface is retained. When returned to seawater and mixed with sperm, the sperm are seen to bind only to the outer surface of the isolated VL (Glabe and Vacquier, 1977).

C. Isolation of Thin Vitelline Layers 1. This method is based on papers by Acevedo-Duncan and Carroll (1986) and Correa and Carroll (1997). All previous work on this subject was reviewed in detail by Weidman and Kay (1986). 2. The egg jelly coats are removed by treatment for 2 min to pH 5.0 seawater, the pH adjusted down with 0.1 N HCl. After readjustment to pH 7 with 0.1 N NaOH, the eggs are settled twice through fresh MFSW. 3. The eggs are fertilized in MFSW with a heavy concentration of sperm. At 20 s after insemination, the eggs are diluted 10-fold into divalent cation-free medium (575 mM NaCl, 10 mM KCl, 10 mM EGTA, 5 mM ethylene glycol, 10 mM Tris pH 8.0, 5 mM benzamidine, and 1 mM 3-amino 1,2,3-triazole, and 50 g/ml soybean trypsin inhibitor). 4. As an alternative to sperm fertilization, the eggs can be activated in ionophore A23187. Ionophore is made up as a 2 mg/ml stock solution in dimethylsulfoxide. The divalent cation-free medium is made 1% in the ionophore stock immediately before addition to the eggs. The final A23187 concentration is 38 M (Glabe and Vacquier, 1978). Each 1 ml of dejellied eggs is resuspended in 10 ml of

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ionophore-divalent cation-free medium. The cortical granules exocytose in unison throughout the entire egg population, causing the VL to elevate at once over the entire egg surface. 5. In either sperm or ionophore activation, the eggs with thin, elevated VLs are washed several times by settling in fresh divalent cation-free medium. The thin VLs are then stripped from the eggs by passing the egg suspension through a Nytex nylon screen of about 100 m pore size. An alternative method is to gently homogenize the eggs with a loose-fitting glass ball homogenizer. With either method, the suspension is checked microscopically to monitor release of the thin VLs. 6. Egg debris is then sedimented by gentle hand centrifugation, visually checking for eggs still in the supernatant. The egg-free supernatant is finally centrifuged at 100 to 300g for 10 min to sediment the thin VLs. The supernatant is discarded and the pellet of thin VLs is resuspended in fresh divalent cation-free medium, stirred with a spatula, and then resedimented. This washing procedure is repeated until the VLs are free of cytoplasm. 7. These thin VLs contain a 350 kDa glycoprotein that has the properties expected of a sperm receptor (Correa and Carroll, 1997).

IV. Isolation of the Cell Surface Complex and the Plasma Membrane–Vitelline Layer (PMVL) Complex from Sea Urchin Eggs A. Introduction The first method to isolate the cell surface complex (CSC) in large quantity was developed in the laboratory of W. J. Lennarz (Detering et al., 1977). The method underwent several revisions and was presented in detail by Kinsey (1986). Since then, several workers (in particular, N. Hirohashi) have modified the procedure, which works especially well for eggs of S. purpuratus. The CSC consists of the cortical granules (CG) (Fig. 1), the plasma membrane (PM), and the vitelline layer (VL). After isolation of the CSC, the CG are dissociated from the PMVL (Kinsey, 1986). The isolation methods (Kinsey, 1986) have been slightly altered to prepare the CSC for identification of a 350 kDa glycoprotein with the characteristics expected of a sperm receptor (Hirohashi and Lennarz, 2001; Ohlendieck et al., 1993). B. Isolation of the Cell Surface Complex 1. All procedures are at 4  C. Eggs are obtained by spawning females into MFSW. Eggs are dejellied by exposure to pH 5.0 for 2 min and then washed 3 times by resuspension, settling, and aspiration of MFSW.

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Fig. 1 Cortical granules from two species of sea urchins. Each is attached to the plasma membrane of the egg, and contains a distinct substructural morphology. Each granule from Strongylocentrotus purpuratus is 1.5 microns in diameter, and from Arbacia punctulata is approximately 1.2 microns. The granules shown from S. purpuratus are labeled with antibodies to a content protein, and show that it is present only in the lamellar region, and not in the lucent homogeneous region.

2. The eggs are washed twice in calcium-magnesium free artificial seawater (CMFSW; 500 mM NaCl, 10 mM KCl, 2.5 mM NaHCO3, 25 mM EGTA, adjusted to pH 8.0 with 1 N NaOH). A 10% vol/vol suspension of the eggs in CMFSW containing a protease inhibitor cocktail is then prepared (CMFSW-PIC). 3. The protease inhibitor cocktail (PIC) can be of two formulations. For example, the Sigma PI (catalog number P-8340) can be diluted 1:500 in CMFSW in addition to final concentrations of 1 mg/ml soybean trypsin inhibitor and 10 mM benzamidine. Alternatively, a stock of leupeptin 1 mg/ml, antipain 2 mg/ ml, benzamidine 10 mg/ml, pepstatin 1 mg/ml can be prepared in 0.5 M EDTA pH 7.5 and added to CMFSW at a 1:500 dilution in addition to 1 mg/ml soybean trypsin inhibitor. 4. The egg suspension in CMFSW-PIC is transferred to a prechilled glass/ Teflon homogenizer and the eggs homogenized slowly, checking continuously with the microscope. It is important not to overhomogenize the CSC, resulting in their fragmentation into small pieces. When >90% of the eggs are lysed, add an additional 10 volumes of CMFSW-PIC, and stir by hand for 2 min, and then centrifuge 800g for 1 min. 5. Discard the supernatant. Carefully remove the upper pellet of white CSC without picking up the bottom yellow pellet of broken eggs, and transfer the CSC to a clean tube. 6. Resuspend CSC in fresh CMFSW-PIC, stir, and repeat the 1 min centrifugation at 800g. After the final wash, the pellet should be fairly pure CSC.

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C. Separation of the Plasma Membrane–Vitelline Layer Complex from the Cortical Granules 1. Remove as much CMFSW-PIC from the CSC pellet as is possible. Add 2 ml ice-cold 1 M sucrose-PIC, allowing the solution to flow gently down the side of the tube. Allow this to wash over the top of the CSC pellet without disturbing the pellet. Pipette oV the sucrose solution. 2. Resuspend the pellet of CSC in 10 vol 1 M sucrose-PIC (the PIC being diluted 1:200 in the 1 M sucrose). Swirl the tube by hand in ice. The cortical granules will detach in 15 to 60 min. Check with the phase contrast microscope every 10 min and gently swirl the tube when checking. 3. When the detachment of CG is complete, centrifuge at 1000g for 20 min. The supernatant containing the cortical granules is removed to a clean tube. 4. Resuspend the final PMVL pellet in CMFSW-PIC and centrifuge 1000g for 1 min. Repeat this wash until the PMVL pellet is clean. 5. Resuspend the final PMVL pellet in CMFSW-PIC. Aliquot and freeze at 80  C. Electron microscopy shows the membrane preparation is quite pure (Kinsey, 1986).

V. The Cytolytic Isolation of the Egg Cortex A. Introduction The cortex of the egg consists of the plasma membrane (PM) and the first 5 m of cytoplasm that is associated with the PM. The cortex is rich in actin and changes in its mechanical properties during fertilization and the cell cycle (reviewed in Vacquier, 1981). Morphogenetic information in the form of specific mRNAs could be diVerentially localized to the cortex. A simple method was discovered where the unfertilized egg, or the zygote, spontaneously contracts so hard as to lyse the cell and roll the cortex oV as an intact structure that is rich in actin (Vacquier and Moy, 1980). Isolation of mRNA from these cortices could be done to characterize what messages are stored in this region. B. Cytolytic Isolation of the Egg Cortex 1. Lytechinus pictus eggs are spawned by injection of adult sea urchins with 0.5 M KCl. The egg jelly coats are dissolved by a 2 min exposure to pH 5.0 seawater, as has been described. 2. Five ml of dejellied eggs is suspended in 100 ml seawater containing 2 mg of pancreatic trypsin. The eggs are stirred gently for 10 min and allowed to settle and the supernatant aspirated away. 3. The eggs are resuspended and then allowed to settle in 50 ml of seawater containing 0.01 M dithiothreitol (DTT), pH 9.1. The eggs are washed in seawater pH 8.0 and allowed to settle. The trypsin/DTT treatment completely removes the egg VL, stripping the surface down to the plasma membrane.

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4. For unfertilized eggs, the isolation medium (IM) is 280 mM NaCl, 350 mM glycine, 20 mM EGTA, 2 mM MgCl2, 42 mM Tris-OH, 5 mM sodium azide, 5 mM benzamidine HCl, 1 mM ATP, pH 7.0. The osmotic pressure of IM is 980 mOsm. Each 1 ml of eggs is suspended in 100 ml of IM at 21  C. Microscopic observation shows that the egg cortex contacts, the egg ruptures, and the cortex peels oV as a single cap-shaped structure. 5. When about 90% of the eggs have lysed, the lysate is poured into 50 ml conical tubes, which are hand-centrifuged 2 min at 216 rpm (radius 17 cm). The pellet of unlysed eggs is removed with a long Pasteur pipette, the contents of the tube mixed gently by inversion and the 2-min centrifugation repeated until no whole eggs remain in the pellet. 6. The intact cortices are then sedimented by a 100g centrifugation for 3 min and washed in fresh IM. The cortices contain the plasma membrane and the cortical granules. 7. To isolate cortices from zygotes, the trypsin/DTT treated eggs are inseminated with a heavy dose of sperm in seawater and then poured into a large petri dish and cultured without agitation. The zygotes are washed by aspiration and addition of fresh seawater (21  C) until the supernatant is clear. 8. At 40 min post-insemination, the zygotes are poured into a 50 ml tube and allowed to settle and the supernatant removed. 9. Each 0.5 ml of zygote pellet is resuspended in 50 ml IM and allowed to stand 30 min at 21  C. The lysed cortices are then collected and washed as previously directed for the unfertilized eggs. Actin is the major component of the zygote cortices (Vacquier and Moy, 1980).

VI. Isolation of Cortical Granules A. Introduction Almost all holoblastically cleaving eggs have cortical granules (CG) bound to the inner surface of the egg plasma membrane. In S. purpuratus, each egg has about 18,000 cortical granules, with an average diameter of 1 m. The CG migrate to the plasma membrane at a late stage in oogenesis (reviewed by Wessel et al., 2001). During normal fertilization, the calcium transient in eggs triggers the CG to fuse with the egg cell membrane. Exocytosis of the CG begins at the point of sperm fusion and radiates around the egg as a circular wave, taking about 30 s to complete. The CG membrane surface area of roughly 57,000 square m is incorporated into the egg cell membrane, also of about 41,000 square m. Thus, during 30 s, the egg’s surface area is roughly doubled (Schroeder, 1979). The extra surface area is extended as elongated surface microvilli and then most of the membrane is retrieved in a massive wave of endocytosis within the first 10 min after completion of exocytosis (Walley et al., 1995). The cortical granule reaction

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is one of the most massive, synchronous examples of exocytosis known. Sea urchin egg cortical granule exocytosis has been studied as a model system for exocytosis and much has been learned. B. Preparation of Cortical Granule Lawns 1. Eggs of S. purpuratus are dejellied and washed extensively in fresh MFSW. Plastic culture dishes, coverslips, or microscope slides are exposed for 5 min to a 1 mg/ml solution of protamine sulfate, or poly-L-lysine, and the surface washed in tap water and air dried. Tissue culture dishes of 5 cm diameter are perfect for this procedure. A 10% suspension of eggs is then applied to the surface. The negatively charged egg VL surfaces bond tightly to the positively charged substrate and the eggs flatten out. 2. The dish is then flooded with CaFSW containing 10 mM EGTA, pH 7.5. However, any solution of approximately 1000 milliosmoles and pH 7 to 8 will work. For example, for low ionic strength, one could use 1 M glycerol with 10 mM EGTA, 10 mM Hepes pH 7.5. After swirling for 2 min, the liquid is flicked out of the dish and the eggs irrigated with a jet of iso-osmotic medium containing 10 mM EGTA, 10 mM Hepes, pH 7.5. The cytoplasm of the eggs is sheared away with the strong jet of calcium chelating medium, leaving the cortices of the eggs firmly bound to the solid support. The negatively charged VL is electrostatically bound to the solid support, the VL is bound to the plasma membrane, and on the inside of the PM is the layer of tightly bound cortical granules (Vacquier, 1975; Fig. 2).

Fig. 2 Isolation of cortical granule lawns. The cortical granules remain attached to the plasma membrane and can further be isolated by shear, or activated by exocytosis by introduction of calcium.

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VII. Isolation of Yolk Platelets Echinoderms are isolecithal animals, meaning that a relatively small amount of yolk (compared to amphibians, avians, or dipterans) is evenly distributed throughout the cells. The major yolk components are packed into distinct membranebound organelles termed yolk platelets or granules. The yolk platelets of sea urchins contain several major molecules made either by the oocyte (YP30, protease) or the adult gut (Major Yolk Protein, or MYP) and reaching the oocyte by selective transport and endocytosis. It should be noted that the overall mass of the yolk platelets does not change during early development (e.g., Harrington and Easton, 1982), calling into question the function of the contents. Several procedures are available for the isolation of yolk platelets. Here, we present a general method for obtaining highly enriched preparations of yolk platelets based on Yokota and Kato (1988) with modifications (Brooks and Wessel, 2002). Additional protocols are available that are variations of the presented protocol, or that take advantage of density gradient media for obtaining purified yolk platelets (e.g., Armant et al., 1986; Harrington and Easton, 1982; Schuel et al., 1975; Unuma et al., 1998). 1. Gametes are obtained by intercoelomic injection of 0.5 M KCl, and the eggs dejellied and washed twice in artificial seawater. 2. To avoid contamination of the yolk preparation by the very abundant cortical granule proteins, the yolk platelet preparation is isolated following fertilization and removal of the fertilization envelope [see Fertilization Envelope section of this chapter for detail]. The preferred approach is to treat eggs with 10 mM dithiothreitol (DTT) to remove the vitelline layer before activation (Epel et al., 1970) so the cortical granule proteins are freely washed away. 3. Eggs are activated by the addition of the calcium ionophore A23187 (10 micrograms/ml) to minimize sperm contamination. 4. Following activation, the cells are kept on ice and washed (by settling) twice with calcium-free seawater and then twice with KCl solution (0.55 M KCl; 1 mM EDTA, pH 7.0). 5. Resuspend the cells in 5 volumes of KCl solution to which a general protease inhibitor cocktail is added and immediately homogenize in a Dounce tissue grinder by hand several times until complete lysis is achieved. 6. Centrifuge the egg homogenate for 4 min at 400g at 4  C, collect the supernatant, and re-centrifuge for 10 min at 2400g. 7. Resuspend the pelleted precipitate in KCl solution and re-centrifuge at 2400g. The final precipitate is the yolk platelet preparation. 8. This preparation is greatly enriched for yolk platelets, as assessed by electron microscopy, with the majority of contamination by mitochondria. For MYP isolation, this preparation is resolved on a SDS-PAGE gel and the 180 kDa MYP band is excised.

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9. For biochemical analysis, we recommend aliquoting the preparation and avoid freeze–thawing, as this results in the loss of a major yolk platelet protein, YP30. The MYP precursor protein can also be isolated from the coelomic fluid of adults (both male and female; see Shyu et al., 1986 and Unuma et al., 1998) and is described in Harrington and Easton (1982). Briefly: 1. Adults (males or females) are opened by cutting the peristomial membrane around Aristotle’s lantern. The perivisceral coelomic fluid is poured out and collected in a beaker on ice to allow the coelomocytes to clot. 2. This clot is removed by decanting the supernatant, and the supernatant is centrifuged for 10 min at 10,000g at 4  C. 3. The supernatant is passed through a Corning disposable sterile syringe filter (0.2 micron; Corning, NY) to remove any residual cellular material, dialyzed at 4  C overnight against several changes of deionized water, and vacuum dried. 4. The relative abundance of MYP is variable in the coelomic fluid but can reach over 75% in well-fed animals. Recently, another constituent of the coelomic fluid of adult sea urchins was identified as an olfactomedin-containing protein, amassin. This protein functions in intercellular adhesion of the coelomic fluid cell population, the coelomocytes, to form the large clots that form in native coelomic fluid (Hillier and Vacquier, 2003).

VIII. Isolation of Mitochondria from Eggs and Embryos Mitochondrial isolation has been used for many studies on the biochemistry of transcriptional regulation, DNA replication, and metabolism. Procedures are prevalent for mitochondrial isolation, and presented here are two of the most general schemes. The first scheme utilizes a sucrose gradient for excellent purification of mitochondria for studies in the biochemistry of DNA replication and transcriptional control (Cantatore et al., 1974; Roberti et al., 1997). The resultant mitochondria are, however, uncoupled from oxidative phosphorylation. A preparation amenable for the studies of oxidative phosphorylation mechanisms will follow, based on the protocol of Selak and Scandella (1987). Protocol for sucrose-gradient purification of mitochondria (Cantatore et al., 1974; Roberti et al., 1997): 1. All steps are performed at 4  C. 2. One ml of packed eggs or embryos is resuspended in 10 mls of 0.25 M sucrose in TEK buVer (100 mM Tris pH 7.6, 1 mM EDTA, 240 mM KCl) and homogenized using a Dounce type glass homogenizer. 3. The homogenate is centrifuged at 600g for 10 min and the supernatant recentrifuged at 2400g for 10 min in order to remove yolk.

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4. The supernatant is again centrifuged at 7500g for 10 min and the pellet containing mitochondria is resuspended in 0.25 M sucrose in TEK buVer (original volume) and centrifuged at 15,000g for 10 min. The enriched mitochondria, at this point, exhibit oxidative phosphorylation but replacement of sucrose with mannitol (see next protocol by Selak and Scandella, 1987) enhances the respirational capabilities of mitochondria. 5. For sucrose-gradient purification of mitochondria, the pellet is resuspended in 0.25 M sucrose/TEK buVer and layered onto a step gradient of 1.0 and 1.5 M sucrose in TEK buVer, and spun in a swinging bucket rotor at 20,000g for 3 h. 6. The mitochondria sediment on top of the 1.5 M sucrose cushion, whereas contaminating yolk settle on top of the 1.0 M sucrose step. 7. The mitochondria are removed by pipetting, diluted into 0.25 M sucrose/ TEK, and repelleted at 15,000g for 10 min. 8. The mitochondria resulting from this protocol are highly purified and have excellent transcription and translational capabilities when an energy source is provided. Protocol for the isolation of mitochondria for studies of respiration (Selak and Scandella, 1987): 1. All steps in the protocol are at 4  C. 2. Washed and dejellied eggs (see preceding text) are resuspended to 10% in isolation medium (300 mM mannitol, 4 mM MgCl2, 4 mM MOPS pH 7.2, 10 mM EGTA, and soybean trypsin inhibitor at 0.5 mg/ml). 3. The cells are homogenized with a Teflon pestle until complete lysis occurs. 4. The lysate is centrifuged twice at 500g for 5 min and then at 12,000g for 10 min. 5. The pellet is resuspended using a loose-fitting Dounce homogenizer in isolation medium at the original volume and recentrifuged at 12,000g for 10 min. 6. The resultant pelleted mitochondria show some contamination by yolk and are held on ice until use. These mitochondria show excellent respirational response to the addition of multiple rounds of ADP and inhibition by addition of oligomycin (blocking the mitochondria F1 ATPase) and the uncouplers CICCP and FCCP.

IX. Isolation of Plasma Membranes and Lipid Rafts from Eggs and Zygotes Understanding fertilization and early development is greatly enhanced by an ability to isolate and analyze the cell surface molecules. Many protocols are available for plasma membrane isolation, and here we present a general isolation

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scheme, as well as the current approach to separating lipid rafts from the membranes of eggs and zygotes. Lipid rafts are microdomains of plasma membranes with a highly ordered lipid phase that partitions and concentrates select proteins involved in cell interactions and signal transduction. Such raft structures are found in many eukaryotic cells, including sperm (see also Chapter 21 by Vacquier and Hirohashi for sperm raft isolation). Membrane raft isolation procedure (Belton et al., 2001): 1. Eggs or zygotes are pelleted at 250g. The seawater is aspirated and the cells resuspended in ice-cold buVer 1 (50 mM Hepes pH 7.4, 250 mM KCl, 5 mM EGTA, 1 mM EDTA, 10 mM NaF, 1 mM sodium vanadate, 1 mM PMSF, and 10 micromolar each of aprotinin, leupeptin, and benzamidine. 2. The cells are then homogenized on ice with a Teflon pestle until 100% lysis is obtained. 3. The homogenate is centrifuged at 150,000g in a swinging bucket rotor for 30 min at 2  C and the supernatant is designated as the cytosolic fraction. 4. The pellet (total particulate fraction) is resuspended in five volumes of buVer 2 (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM EGTA, 1 mM EDTA, 2% Triton X-100, 10 mM NaF, 1 mM sodium vanadate, 1 mM PMSF and 10 micromolar each of aprotinin, leupeptin, and benzamidine), and the pellet is lightly homogenized until it appears completely dissolved. It is then incubated for 15 min more on ice. 5. The dissolved pellet is then centrifuged at 150,000g for 30 min at 2  C to separate the Triton X-100 insoluble and soluble fractions. For analysis of the insoluble fraction at this point, use SDS sample buVer containing 10 mM NaF and 1 mM sodium vanadate to inhibit phosphatase activity. 6. The Triton X-100-insoluble fraction is resuspended in an equal volume of 80% sucrose in buVer 2 to give a final sucrose concentration of 40%. 7. Three to 4 mls of this fraction is layered into an ultracentrifuge tube designed for sucrose gradients, and overlayed with 6 mls of 30% sucrose buVer 2, then with 6 mls of 5% sucrose in buVer 2. The samples are centrifuged at 250,000g for 16 h at 2  C and 1 ml fractions are collected from the top of the gradient. 8. Prior to SDS analysis, aliquots of each fraction are dialyzed against cold buVer 2 without Triton X-100. 9. To disrupt the lipid rafts by removal of cholesterol, treat with the cholesterol-binding agent, methyl-
-cyclodextrin (M
CD) dissolved in buVer 2 with protease and phosphatase inhibitors, but without Triton X-100, at 16  C for 1 h. The levels of M
CD used for extraction have been examined (0–50 mM) and are documented (Belton et al., 2001). 10. The M
CD soluble and insoluble fractions are separated by centrifugation at 21,000g for 1 h at 4  C. Several phosphotyrosine-containing proteins are known to be present in the rafts, including the protein kinase Src, and are released (M
CD-soluble) following cholesterol treatment.

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Several other plasma membrane isolation procedures for eggs and embryos are available. Readers are especially encouraged to explore the detailed protocols of Kinsey (1986) and modifications given in Giusti et al. (1997) as well as the isolation procedure and analysis of membranes from embryos by Cestelli et al. (1975).

X. Isolation of Microsomes Containing the Endoplasmic Reticulum The endoplasmic reticulum of eggs contains calcium stores required for egg activation. At fertilization, signal transduction events leading to the formation of inositol tris-phosphate stimulates calcium release from the endoplasmic reticulum. Procedures have been developed to enrich for the endoplasmic reticulum in functional form i.e., uptake and release of calcium. We document here the procedure for isolation of the microsomes, which, by enzymatic and microscopic analysis, are enriched in the endoplasmic reticulum (Oberdorf et al., 1986). Procedure (All steps are performed at 4  C.): 1. Eggs are washed three times in calcium-free seawater and resuspended in 10 volumes of buVer B (0.5 M KCl, 10 mM MgCl2, 10 mM 2-[N-morpholino]propane sulfonic acid, 10 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine, pH 7.0). 2. The eggs are then homogenized with a Teflon homogenizer and sequentially pelleted in a fixed-angle rotor as follows: 200g for 3 min (egg cortex); 3000g for 20 min (yolk); 15,000g for 20 min (mitochondria). 3. The 15,000g supernatant is spun at 100,000g for 30 min to pellet the microsomes. The microsomes pack loosely over the more dense, aggregated pellet. 4. The microsomes are carefully separated from the lower pellet and layered on a 0.3 M sucrose, 0.7 M glycine pH 7.0 cushion and the pellets from a 100,000g spin for 30 min were stored at 80  C. This protocol is modified from an earlier procedure (Inoue and Yoshioka, 1982).

XI. Nuclear Isolation Procedures Several procedures are available for isolating nuclei from echinoderm oocytes, eggs and, embryos, and the choice of procedures depends on the application. Two major applications of isolated nuclei in sea urchins are (1) nuclear run-on (or runoV) experiments to measure as closely as possible the in vivo transcriptional activity of specific genes in select cell-types, and (2) biochemical analysis of transcription factors from nuclear extracts. Both applications and procedures are well documented, and the reader will be referred to other sources for detail. Transcription of RNA in isolated nuclei is used to mimic the transcriptional activity in intact cells. Chromatin is maintained in its native state following

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nuclear isolation and the newly synthesized transcripts reflect the activity of that nucleus in the cell. By isolation, though, one is able to readily label the newly synthesized transcripts and measure their abundance in relative units. In one reaction, an investigator can measure the transcriptional activity of the cell for any one of the thousands of genes that may be transcribed in the cell at that time. We found MarzluV and Huang (1984) the most useful and comprehensive source for the entire procedure of nuclear isolation and in vitro transcription. Much of the development of the procedures by these investigators was using sea urchin embryos and the reference given specifically addresses sea urchin nuclear isolation. Procedures for isolating nuclei for biochemical extracts of trans-factors is given in Chapter 26 by CoVman and Yuh in this volume, and isolation of pronuclei from both eggs and sperm are given in Poccia and Green (1986). Extensive detail is given in the isolation, extraction, and application of nuclei in those sources and will not be repeated here. Isolation procedures are also available for germinal vesicles from oocytes of starfish (Chiba et al., 1995). The procedure given below (Nemoto et al., 1992) relies on first disrupting the actin–cytoskeletal matrix, which enables the germinal vesicles to be released intact by the cells. Procedure: 1. Starfish oocytes denuded of their extracellular layers (see Chapter 3 by Foltz et al. for procedures) were resuspended in calcium-free seawater containing 10 micrograms/ml cytochalasin B (stock 2 mg/ml in DMSO). 2. Approximately 5 mls of the treated oocytes are collected by sedimentation and gently layered on a discontinuous sucrose gradient in a 50 ml tube containing diVerent sucrose (1 M) mixtures with calcium-free seawater (CaFSW). From top to bottom, the gradient steps are: (1) 3 parts sucrose:7 parts CaFSW (7 mls); (2) 4 parts sucrose:6 parts CaFSW (11 mls); (3) 5 parts sucrose; 5 parts CaFSW (7 mls); and (4) 1.2 M sucrose (5 mls). 3. The preparations are then centrifuged in a swinging bucket rotor (5500g for 20 min at 4  C). The oocytes sediment to the top of step 4 whereas the germinal vesicles sediment to the top of step 2. The time and speed of centrifugation will vary for diVerent species—the values given are for the Japanese starfish Asterias amurensis and need to be adjusted for use in other species. 4. The germinal vesicles are gently removed by pipetting and are washed three times by centrifugation (1000g for 3 min) in cold washing medium (0.2 M sucrose, 0.3 M KCl, 5 mM MgCl2, and 10 mM Tris pH 7.5). 5. The final pellet is kept in an ice bath or stored at 80  C until use.

XII. Removal and Isolation of the Fertilization Envelope The fertilization envelope forms within 60 s of insemination, and becomes stabilized by chemical cross-linking within a few minutes. The envelope forms

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largely by the secreted contents of the cortical granules mixing with the vitelline layer scaVolding as it lifts oV the plasma membrane surface by hydration forces. This envelope protects the embryo from mechanical and biochemical insults until hatching but may also thwart investigators wishing to access the blastomeres for experimental manipulation. Several protocols have been developed to remove the envelope following fertilization or to prevent it from forming in the first place (Fig. 3). Other protocols have been established to isolate the envelope, or various parts of the envelope and its constituents (see preceding text for vitelline layer; see Weidman and Kay (1986) for detailed description of biochemical approaches for isolating cortical granule content proteins, and fertilization envelope intermediates; Fig. 4). Three approaches will be documented here to either (1) remove the envelope at some point following fertilization; (2) isolate the envelope; or (3) prevent the envelope from forming. A. Removal of the Envelope during Embryogenesis For access to blastomeres early in development, the fertilization envelope may have to be removed. This can be performed shortly after fertilization, though if prolonged culture of the embryos is needed before experimentation, it is often advisable to keep the envelope on the embryo until just prior to experimentation. The envelope reduces the stickiness of the exposed hyalin layer and resultant clumping of the embryos. The stabilization of the fertilization envelope is due to chemical cross-linking of tyrosine residues by ovoperoxidase. If one blocks the activity of ovoperoxidase, the envelope still forms normally, it just does not stabilize and is thus easy to remove at any point following fertilization.

Fig. 3 Fertilization envelope isolation. The envelopes are stripped oV the early embryo by first inhibiting the cross-linking reaction of ovoperoxidase, and then pouring the embryos through a nylon mesh to shear the envelopes oV the cell. FE, fertilization envelope; PN, male (left) and female (right) pronuclei. Bar ¼ 25 microns.

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Fig. 4 The isolated fertilization envelope from Strongylocentrotus purpuratus contains a small population of abundant proteins as seen by SDS-PAGE.

To inhibit activity of ovoperoxidase, two popular reagents are used (Weidman and Kay, 1986): (a) 2 mM 3-amino-1,2,3-triazole in seawater (from a 1 M stock in deionized water), and (b) 10 mM para-aminobenzoic acid in seawater (make fresh before using, keep out of direct light, and pH the seawater to pH 8.0 following dissolution of the pABA). Eggs should be washed once in the reagent and then fertilized and cultured in the inhibitor. Embryos develop normally to larvae in either reagent condition. When envelopes need to be removed, pass the embryos through Nitex nylon mesh. Sizes of Nitex used depend on the embryo. The strategy is to choose the size through which the embryo will pass, but which the envelope would need to deform. This deformation is usually suYcient to strip the envelope oV the embryo and both it and the embryo will then pass through the Nitex. If too large a mesh is selected, the embryos pass through unaltered, whereas if too small a mesh is used, the embryos collect on the top of the Nitex and the envelopes are not removed. Representative Nitex sizes are usually as follows: for S. purpuratus, 64 microns, for L. variegatus, 80 microns. A convenient apparatus for the Nitex mesh is a plastic beaker that has had its bottom cut oV. The size of the beaker depends on the volume of embryos to be processed. Usually, a 100 to 400 ml beaker is suYcient for a wide range of applications. Place the Nitex flat over the top of the beaker, and aYx a rubber band around the rim to hold the Nitex snugly in place. Pour the treated embryos

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through the Nitex and collect with another beaker. Do not swirl the embryos because this will increase shear and lysis. Monitor envelope removal on the microscope and repeat once or twice if necessary. B. Isolation of Fertilization Envelopes Following removal of the envelopes from embryos (see #1 in preceding text), both fractions can be collected by diVerential centrifugation. The embryos can be isolated by low-speed spins (500g or with a hand centrifuge for 2 min). Repeat until eVectively all the embryos are removed from the supernatant. To collect the fertilization envelopes, spin at high speed on a clinical centrifuge (4 – 5000g for 5 min). The pelleted envelopes should appear white on top of any residual embryos or debris, and can usually be removed by gentle resuspension with a Pasteur pipette without disturbing the contaminating debris. C. Prevent Fertilization Envelopes from Forming An eVective way to prevent envelopes from forming is to disrupt the vitelline layer needed to scaVold the cortical granule contents. A simple and eVective method to disrupt the vitelline layer is by treatment with pH 8.0 seawater containing 10 mM dithiothreitol (DTT; Epel et al., 1970). Simply resuspend the embryos in the DTT for 10 min and then wash the eggs several times in seawater. Removal of the egg jelly is first required for eVective DTT treatment (see preceding text for acid treatment of eggs to remove jelly).

XIII. Isolation of Cilia from Embryos Deciliation is accomplished by immersing embryos briefly in hypertonic medium, as used originally in Auclair and Siegel (1966; see also Stephens, 1986, for background, and more recent applications by Casano et al., 1998). Sea urchin embryos are best deciliated by 2X seawater (1.0 M NaCl). 1. To make 2X seawater, add an additional 0.5 M NaCl (29.2 gm/l) to normal seawater. 2. To deciliate embryos, first pellet the embryos gently (1000g for 2 min), remove the supernatant, and then add 10 volumes of 2X seawater at the normal growth temperature of the embryo. Gently resuspend embryos quickly and completely, and keep embryos resuspended during the deciliation process. It is imperative that the embryos be handled gently to minimize lysis. Even if only a small percent of the embryos lyse during the procedure, abundant cytoplasmic components, e.g., yolk, will contaminate the cilia preparation. Monitor deciliation continuously with a phase microscope, leaving embryos in hypertonic medium no longer than is necessary, usually about 2 min.

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3. Sediment the deciliated embryos by low-speed centrifugation (1000g for 2 min) and remove the supernatant carefully with a pipette or by careful decanting. Repeat with increased centrifugation if residual embryos remain. 4. For examination of regenerating cilia in the embryos, resuspend the embryos in original growth conditions. Embryos will regenerate their cilia rapidly and will develop normally, even after multiple deciliations (see Stephens, 1986). 5. For recovery of the cilia, pellet them at 10,000g for 10 min at 4  C. Notes: 1. Prior to deciliation, it is sometimes helpful to first wash the embryos through nylon mesh, collecting the embryos while washing through any contaminants. This wash will minimize any microbial contamination or residual lysed embryos in the cilia preparation. 2. All cells of the swimming blastula generate and then, following retraction at mitosis, regenerate their cilia. Cilia growth ceases in ingressing mesechyme cells, e.g., primary mesenchyme cells. Cilia are also lost from invaginating endoderm although the foregut retains significant cilia function.

XIV. Isolation of Extracellular Matrices from Embryos and Larvae The embryo has many extracellular matrix molecules divided among its two extracellular environments. The inside of the embryo contains a blastocoel, composed of a mixture of ECM molecules, through which cells of the developing embryo will migrate and extend cellular processes. Surrounding the blastocoelar matrix, and immediately underlying the epithelium of the embryonic cells, is the basal lamina (sometimes erroneously referred to as a basement membrane or basal membrane, though the structure has no lipid bilayer, and the terms are easily confused with the basal membrane domain of the plasma membrane of an epithelial cell). The basal lamina is a concentrated extracellular matrix, is readily apparent in the electron microscope, and has distinct molecular composition. The outside of the embryo (apical aspect of the epithelial cells) is surrounded by an extracellular matrix distinct from the blastocoel environment. The apical, or extraembryonic matrix, consists of an apical lamina immediately adjacent to the epithelial cells, surrounded by a hyaline layer on the outermost surface. Each of the extracellular matrix domains can be isolated and such procedures will be documented here. A. Isolation of Hyalin Hyalin is the major protein of the hyaline layer, the outermost layer of the embryo. Hyalin is made from two sources: maternal—the developing oocytes package the protein into cortical granules, and release the hyalin at fertilization;

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and zygotic—new hyalin mRNA is synthesized in most cells during gastrulation, and is secreted to form a new hyalin extracellular matrix pool (Wessel et al., 1998). It appears that the mRNA used for translation in embryos is derived from the same gene as the oocyte version, and both maternal and zygotic forms of the mRNA and protein are indistinguishable. Hyalin appears to be a calcium-binding protein and removal of calcium solubilizes hyalin, while addition of calcium to solubilized hyalin re-precipitates, or gelates, the hyalin (Kane, 1973). Hyalin is most readily isolated one hour following fertilization, though the same procedure can be used to isolate hyalin from any stage of development. 1. If hyalin is to be removed prior to hatching, the fertilization envelope must first be removed (see section on fertilization envelope isolation). This is accomplished by fertilizing eggs in reagents that block the activity of ovoperoxidasemediate crosslinking of the fertilization envelope, e.g., 10 mM para-aminobenzoic acid (note that the seawater must be re-pHed following addition of pABA or 2 mM 3-aminotriazole (Showman and Foeder, 1979; Weidman and Kay, 1986). The envelope will still form; it will just not be stabilized. One hour following fertilization in either of the above reagents, pass the zygotes through Nitex mesh (see fertilization envelope isolation procedure for details of sizes). Remove embryos by settling or gentle centrifugation; use caution as they are now very sticky with their exposed hyalin layers. 2. Resuspend the embryos in 10 volumes of hyalin extraction medium (see Appendix) at the temperature of normal embryo culture. Gently swirl embryos and keep in suspension for 10 min (caution: do not lyse embryos, because the hyalin preparation will become contaminated with abundant cytoplasmic components, e.g., yolk). 3. Remove embryos by gentle centrifugation (1000g for 2 min) and collect the supernatant. (Note: if the embryos are to be recultured, first wash them once in seawater and then replace them into culture conditions more sparse than normal—no more than 0.2%—to minimize the clumping introduced by fertilization envelope removal.) Repeat centrifugation as necessary to remove all embryos and cells. Recentrifuge the supernatant at higher force (10,000g for 10 min) and again collect the supernatant containing the solubilized hyalin and place on ice. 4. Add calcium (from a 1 M stock of CaCl2) gradually with gentle stirring at 4  C to a final concentration of 20 mM and continue stirring for 30 min. 5. Centrifuge the precipitated hyalin for 10 min at 10,000g for 10 min, and decant the supernatant. Hyalin appears as a translucent, gelatinous pellet. 6. If significant purity of hyalin is needed, or if the hyalin preparation is discolored by contamination, the process of calcium-free solubilization and calcium-mediated precipitation is employed. To re-solubilize the precipitated hyalin, add calcium- and magnesium-free seawater to which 2 mM EDTA (CMFE) has been added. Use the original volume of CMFE used to solubilize hyalin. Gently swirl the hyalin resuspension for 30 min, and recentrifuge at 10,000g for 10 min.

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7. Repeat the calcium-mediated precipitation process as above. Continual calcium-precipitation/EDTA-solubilization does help purify the hyalin but at a significant expense of yield. Sometimes it is beneficial to dialyze the solubilized hyalin against CMFE for several hours to aid in solubilization and to remove additional contaminants. It is best to start the purification process with as little lysis of embryos as possible. B. The Apical Lamina The apical lamina (AL) is a fibrous extracellular layer intimately associated with the clear hyaline layer (HL) that forms on the sea urchin egg plasma membrane after cortical granule exocytosis. The HL proteins can be solubilized from the egg surface by treatment with 1.1 M glycine, leaving the fibrous AL bound to the egg surface (Hall and Vacquier, 1982). The AL is composed of three major glycoproteins of 175, 145, and 110 kDa. Uronic acid, sialic acid, and collagen are not found in the AL, but sulfate is present. Unlike hyalin, the soluble AL glycoproteins do not precipitate in the presence of calcium. The AL does not change in composition between the zygote and the hatched blastula stage. The AL is extremely sticky and diYcult to work with as an intact layer. It is hypothesized that the AL plays a role in blastomere adhesion (Hall and Vacquier, 1982). The cloning of the AL proteins showed them to contain EGF repeats and they were named ‘‘fibropillins.’’ At the blastula stage, fibropillins become organized into fibers on the surface of the embryo (Bisgrove and RaV, 1993; Bisgrove et al., 1991). Phylogenetic studies indicate that the fibropillins of the AL have been conserved for at least 250 million years (Bisgrove et al., 1995). Antibodies to one fibropillin immunoprecipitate a complex of all three AL fibropillins. The AL fibropillins appear to be synthesized throughout cleavage to the hatching blastula stage (Burke et al., 1998). Apextrin, another AL component, has been identified in the AL of the direct developing sea urchin Heliocidaris erythrogramma (Haag et al., 1999). The AL isolation method described in the following text is taken from Hall and Vacquier (1982). 1. Hyaline layer-apical lamina (HL-AL) complexes are isolated from eggs which have been treated with 10 mM dithiothreitol (DTT), pH 9.1, for 10 min. This prevents the vitelline envelope from elevating and forming the fertilization envelope (Epel et al., 1970). The DTT-treated eggs are fertilized and cultured at 15  C under normal conditions. 2. The embryos are concentrated to 10% vol/vol, packed in ice, and 20% Triton X-100 added to a final concentration of 1%. 3. The detergent-extracted embryos are then pressurized in a nitrogen cavitation bomb to 1200 psi. (Yeda Press, a Parr Bomb, or a French Press will accomplish the disruption.) The exact pressure has to be worked out empirically for each species and each apparatus. 4. The embryos are passed through the small orifice of the pressure release valve and are lysed and collected in a beaker. The HL-AL complexes are

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sedimented by centrifugation at 180g for 1 min. The pellet is then resuspended in 50 vol ice-cold filtered seawater with 1% Triton X-100. The pellet is resuspended by vigorous pipetting. The HL-AL complexes are then sedimented again. This wash is repeated a total of three times. 5. To isolate the AL fibropillins, HL-AL complexes are suspended in 30 vol of ice-cold 1.1 M glycine, 2 mM EGTA adjusted to pH 8.0. The material is allowed to stand for 30 min on ice with gentle mixing. The glycine extraction solubilizes the hyalin protein, while the fibropillin containing fibrous AL remain insoluble. The insoluble AL can be washed twice in the glycine buVer to rid them of contaminating proteins. The AL are greatly enriched in the 175, 245, and 110 kDa fibropillin proteins. 6. The hyalin protein can be purified by preparing a 10,000g supernatant of the glycine extracted complexes. The supernatant is dialyzed against 20 mM CaCl2, 20 mM Tris-HCl, pH 8.0. The calcium–proteinate precipitate is then recovered by low-speed centrifugation. 7. Living embryos can also be extracted in the glycine buVer. The result is that the hyalin protein goes into solution, leaving the fibrous AL on the surface of the embryo, where it is clearly visible by scanning EM.

C. The Basal Lamina/Blastocoel Matrix Two general methods are used to isolate the blastocoelar extracellular matrix. One is used to obtain an intact blastocoel with mesenchyme cells intact. These preparations are amenable to cellular manipulation and observation in situ. The second approach is a biochemical preparation. Both will be documented here. For intact blastocoel preparations, the method of Harkey and Whiteley (1980) is used. This preparation is used to isolate intact ECM with mesenchyme cells in their native environment, but in it the cells and ECM can be readily manipulated. This procedure was also used for mesenchyme cell isolation but now other, larger scale and easier protocols are available (see Chapter 14). The basis of this protocol is rapid dissociation of embryos with minimal mechanical shearing. The basal lamina bags are very delicate, and normal procedures to strip epithelial cells from embryos are suYciently harsh as to fragment the bags. The best dissociation occurs at early gastrula stages, following primary mesenchyme cell ingression, and about 1/4 invagination of the archenteron. The procedures were developed for a variety of embryo types—Strongylocentrotus purpuratus, S. droebachiensis, and the sand dollar, Dendraster excentricus—and minor adjustment of times and centrifugation forces may be necessary for other species.

1. Procedure Pellet embryos by hand centrifugation or settling and gently wash 3 times in 15 volumes of CMFSW, twice in 5 to 15 volumes of DM, and then resuspend in BIM.

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These steps are all carried out on ice. Monitor the dissociation process microscopically, especially the second DM wash, as one wants to get loosening of the epithelial cells in DM so that when they are washed into BIM, complete dissociation occurs, with occasional gentle mixing to maximize dissociation. Bag isolation procedure A: This procedure exploits a diVerence in sedimentation of bags and dissociated single cells. 1. Bags are partially purified by 4 cycles of sedimentation at 250g for 2 min, followed by resuspension in 20 volumes of BIM. 2. The resuspension is layered as 10 ml aliquots over several sucrose step gradients, each consisting of 10 ml steps of 30% sucrose stock–70% BIM, 50% sucrose stock–50% BIM, and a sucrose stock cushion. 3. The preparation is centrifuged 3 min at 650 g in a swinging bucket rotor. The vast majority of bags with their constituent cells inside are found on the 50% step. Bag isolation procedure B: This approach utilizes the diVerence in densities between bags and dissociated epithelial cells. Their densities were estimated from a linear Percoll gradient: dissociated cells from early gastrulae band at 22.64% Percoll (1.062 g/ml), while bags banded loosely with a peak at 16.73% Percoll (1.054 g/ml). A range of densities is seen for bags that are empty (lighter) versus bags with many adherent cells. 1. A freshly dissociated cell suspension is settled on ice and resuspended with BIM and Percoll stock making the suspension 30 to 40% cells and 19% Percoll by volume. 2. The preparation is centrifuged for 20 min at 650 g in a swinging bucket rotor, with a BIM overlay and a cushion of sucrose stock. To reduce mixing during deceleration, use of small diameter centrifuge tubes is preferable. 3. Bags accumulate at the BIM interface. These are collected by pipette, resuspended in BIM, and further purified on a sucrose gradient, as in Procedure A.

2. Blastocoel Bag Isolation Reagents CMFSW (calcium- and magnesium-free seawater): see Appendix, containing 100 micromolar EDTA, pH 8.0 DM (dissociation medium): 1 M glycine, 100 micromolar EDTA, pH 8.0 BIM: (bag isolation medium): 40% CMFSW, 40% 1 M dextrose, 20% distilled water, pH 8.0. Percoll stock: 84% Percoll (Pharmacia Fine Chemicals), 16% 5 CMFSW Sucrose stock: 1 M sucrose, 100 micromolar EDTA, 1 mM Tris, pH 8.0 An alternative method has been use for large-scale biochemical preparations of extracellular matrix (Wessel et al., 1984). These preparations have been used for

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biochemical analysis, as a substrate for cell attachment, and as a source to generate antibodies. 1. Embryos are pelleted gently, and then resuspended in ice-cold extraction buVer (0.1% Triton X-100, 10 mM NaHCO3, protease inhibitors (especially 0.01% phenylmethylsulfonylfluoride), and 15 micrograms per ml of DNase. The combination of low ionic strength and detergent removes cellular materials and enriches for the inherently insoluble extracellular matrix. Extract for 15 min while keeping the embryos in suspension on a rotator or rocking table at 4  C. 2. The matrix remaining can either be pelleted at 15,000g for 30 min at 4  C or collected by running the extraction through 80 micron Nitex mesh. 3. Repeat extractions 2 to 3 times until cellular debris is removed. 4. Enhanced preparations can be obtained by first removing the hyalin layer (see preceding text) and the epithelium (see chapter 14 by McClay for cell dissociation) prior to the extraction. Notes: 1. The older the embryos, the more stable the extracellular matrix. Usually, embryos prior to gastrulation are diYcult to isolate by this procedure, but following gastrulation, the extracellular matrix becomes more robust and is stable to extraction. A trade-oV though is increased contamination by mesenchyme cells within the blastocoel. 2. If the extracellular matrix preparation is to be used for live cell work, care must be taken to remove the detergent in the extraction buVer. If the preparation is to be used as a substrate plated on plastic dishes, it can be washed repeatedly with seawater. Other applications may require extensive dialysis against seawater. 3. These preparations have been successfully used to study invasion of metastatic cells from mammals (Livant et al., 1995). To prepare the extracellular matrix for incubation with live cells, wash the preparation several times by pelleting in sterile seawater, followed by washing in culture media.

XV. Isolation of Sea Urchin Larval Skeletons The skeleton of larvae is a prominent structure and is intensely birefringent. Significant enrichments of the spicule can be made by first lysing the larvae in hypotonic buVer, and then repeatedly washing the lysate in detergent-containing buVers. The major contaminant in such preparations is the basal lamina/blastocoel extracellular matrix, which is diYcult to solubilize. Urea helps in removal of the contaminants (see Benson et al., 1986), but a more eVective way is to briefly wash the enriched skeletons with alkaline sodium hypochlorite. The method is rapid, eVective, and results in skeletal preparations relatively clean of contaminating proteins. The protocol for skeleton isolation will be given from the detailed analysis and utilization of these preparations in Benson et al. (1986). These

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preparations were instrumental in the identification of the spicule matrix proteins, e.g., SM50 and SM 30. 1. Collect prism stage embryos or larvae and wash once in calcium- and magnesium-free seawater and twice in ice-cold 1.5 M glucose. Pellet the larvae. 2. Resuspend the larvae in 10 mM Tris pH 7.4 containing broad spectrum protease inhibitors and lightly resuspend in a loose-fitting Dounce homogenizer. Allow the lysate to sit on ice for 10 min and centrifuge for 3 min at 800g in plastic tubes. Throughout the procedure, use plastic vessels wherever possible because low yields have been seen when preparations are made in glass. 3. Discard the supernatant and repeat the homogenization procedure. 4. Resuspend the pellet in 10 volumes of 2% Triton X-100, 4% sodium deoxycholate, 20 mM Tris pH 7.4, and homogenize in a loose-fitting Dounce homogenizer. 5. Centrifuge (800g for 3 min) the suspension and repeat extraction of the pellet 4 to 5 times until most of the cellular debris is removed and the pellet is light brown to oV-white. 6. Resuspend again in the same buVer and, while lightly homogenizing again, add 5 original embryo volumes of cold 5% sodium hypochlorite, 10 mM Tris pH 8.0. Continue homogenization for 30 to 60 s. As the preparation turns clear white, quickly centrifuge at 800g for 1 min and wash by resuspending the skeletons in cold 2% hypochlorite 10 mM Tris pH 8.0. 7. Centrifuge again as previously directed and wash the skeletons 2 to 3 times with ice-cold distilled water. 8. The isolated skeletons can be demineralized by resuspending the waterwashed pellet in 50 mM EDTA pH 8.0 or in 0.1 N acetic acid. Some residue usually remains after demineralization and is usually attributed to dust and salt crystals, though some insoluble skeleton debris may be present as well. Remove the debris by centrifugation at 1000g for 2 min. 9. The soluble skeleton matrix is then dialyzed against several changes of cold distilled water, lyophilized, and stored frozen. The lyophilized powder resuspends well in aqueous solutions. Skeletons may also be analyzed and isolated from cultures in which micromeres have been allowed to develop. See chapter 14 for further information.

XVI. Resources for the Isolation of Additional Organelles A. Mitotic Spindles Because of the great synchrony in development of early sea urchin embryos, embryos will progress through the cell cycle en masse, and protocols have been developed to isolate mitotic spindles at diVerent stages of mitosis. In some

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protocols, these spindles are capable of progressing through the cell cycle— exhibiting pole–pole elongation similar to that observed in vivo. Readers are referred especially to the following references: Palazzo et al., 1991; Rebhun and Palazzo, 1988; reviews on the subject by Kuriyama, 1986; Silver, 1986. References Acevedo-Duncan, M., and Carroll, E. J. (1986). Immunological evidence that a 305 kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor. Gamete Res. 15, 337–359. Alves, A. P., Mulloy, B., Moy, G. W., Vacquier, V. D., and Moura˜ o, P. A. S. (1998). Females of the sea urchin Strongylocentrotus purpuratus diVer in the structures of their egg jelly sulfated fucans. Glycobiology 8, 939–946. AminoV, D. (1961). Methods for quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem. J. 81, 384–392. Armant, D. R., Carson, D. D., Decker, G. L., Welply, J. K., and Lennarz, W. J. (1986). Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata. Dev. Biol. 113, 342–355. Auclair, W., and Siegel, B. W. (1966). Cilia regeneration in the sea urchin embryo: Evidence for a pool of ciliary proteins. Science 154, 913–915. Belton, R. J., Adams, N. L., and Foltz, K. R. (2001). Isolation and characterization of sea urchin egg lipid rafts and thier possible function during fertilization. Mol. Reprod. Dev. 59, 294–305. Benson, S. C., Jones, E. M., Benson, N. C., and Wilt, F. (1986). Morphology of the organic matrix of the spicule of sea urchin larvae. Exp. Cell Res. 148, 249–253. Benson, S. C., Benson, N. C., and Wilt, F. (1986). The organic matrix of the skeletal spicule of sea urchin embryos. J. Cell Biol. 102, 1878–1886. Bisgrove, B. W., Andrews, M. E., and RaV, R. A. (1991). Fibropillins, products of an EGF repeat containing gene, form a unique extracellular matrix structure that surrounds the sea urchin embryo. Dev. Biol. 146, 89–99. Bisgrove, B. W., and RaV, R. A. (1993). The SpEGFIII gene encodes a member of the fibropillins: EGF repeat-containing proteins that form the apical lamina of the sea urchin embryo. Dev. Biol. 157, 526–538. Bisgrove, B. W., Andrews, M. E., and RaV, R. A. (1995). Evolution of the fibropillin gene family and patterns of fibropillin gene expression in sea urchin phylogeny. J. Mol. Evol. 41, 34–45. Bonnell, B. S., and Chandler, D. E. (1990). Visualization of the Lytechinus pictus egg jelly coat in platinum replicas. J. Struct. Biol. 105, 123–132. Bonnell, B. S., Keller, S. H., Vacquier, V. D., and Chandler, D. E. (1994). The sea urchin egg jelly layer consists of globular glycoproteins bound to a fibrous fucan superstructure. Dev. Biol. 162, 313–324. Brooks, J. M., and Wessel, G. M. (2002). The major yolk protein in sea urchins is a transferrin-like, iron binding protein. Dev. Biol. 245, 1–12. Burke, R. D., Lail, M., and Nakajima, Y. (1998). The apical lamina and its role in cell adhesion in sea urchin embryos. Cell Adhes. Commun. 5, 97–108. Cantatore, P., Nicotra, A., Loria, P., and Saccone, C. (1974). RNA synthesis in isolated mitochondria from sea urchin embryos. Cell DiV. 3, 45–53. Casano, C., Roccheri, M. C., Onorato, K., Cascino, D., and Gianguzzi, F. (1998). Deciliation: A stressful event for Paracentrotus lividus embryos. Biochem. Biophys. Res. Comm. 248, 628–634. Cestelli, A. M., Albeggiani, G., Allotta, S., and Vittorelli, M. L. (1975). Isolation of the plasma membrane from sea urchin embryos. Cell DiV. 4, 305–311. Chandler, D. E., and Heuser, J. E. (1980). The vitelline layer of the sea urchin egg and its modification during fertilization. J. Cell Biol. 84, 618–632. Chiba, K., Nakano, T., and Hoshi, M. (1995). Induction of germinal vesicle breakdown in a cell-free preparation from starfish oocytes. Dev. Biol. 205, 217–223.

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Gary M. Wessel and Victor D. Vacquier Correa, L. M., and Carroll, E. J. (1997). Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus. Develop. Growth & DiVer. 39, 69–85. Darszon, A., Beltran, C., Felix, R., Nishigaki, T., and Trevino, C. L. (2001). Ion transport in sperm signaling. Dev. Biol. 240, 1–14. Detering, N. K., Decker, G. L., Schmell, E. D., and Lennarz, W. J. (1977). Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs. J. Cell Biol. 75, 899–914. Dubois, M., Gilies, J. A., Hamilton, J. K., Robers, P. A., and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Epel, D., Weaver, A. M., and Mazia, D. (1970). Methods for removal of the vitelline membrane of sea urchin eggs. 1. Use of dithiothreitol (Cleland’s reagent). Exp. Cell Res. 61, 64–68. Farndale, R. W., Buttle, D. J., and Barrett, A. J. (1986). Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 883, 173–177. Gache, C., Niman, H. L., and Vacquier, V. D. (1983). Monoclonal antibodies to the sea urchin egg vitelline layer inhibit fertilization by blocking sperm adhesion. Exp. Cell Res. 147, 75–84. Garbers, D. L. (1989). Molecular basis of fertilization. Annu. Rev. Biochem. 58, 719–742. Giusti, A. F., Hoang, K. M., and Foltz, K. R. (1997). Surface location of the sea urchin egg receptor for sperm. Dev. Biol. 184, 10–24. Glabe, C. G., and Vacquier, V. D. (1977). Isolation and characterization of the vitelline layer of sea urchin eggs. J. Cell Biol. 75, 410–421. Glabe, C. G., and Vacquier, V. D. (1978). Egg surface glycoprotein receptor for sea urchin sperm binding. Proc. Natl. Acad. Sci. USA 75, 881–885. Gonzales, P., and Lessios, H. A. (1999). Evolution of sea urchin retroviral-like (SURL) elements: Evidence from 40 echinoid species. Mol. Biol. Evol. 16, 938–952. Haag, E. S., Sly, B. J., Andrews, M. E., and RaV, R. A. (1999). Apextrin, a novel extracellular protein associated with larval ectoderm evolution in Heliocidaris erythrogramma. Dev. Biol. 211, 77–87. Haley, S. A., and Wessel, G. M. (1999). The cortical granule serine protease CGSP1 of the sea urchin, Strongylocentrotus purpuratus, is autocatalytic and contains a low density lipoprotein receptor-like domain. Dev. Biol. 211, 1–10. Hall, H. G., and Vacquier, V. D. (1982). The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer. Dev. Biol. 89, 168–178. Harkey, M. A., and Whitely, A. H. (1980). Isolation, culture, and diVerentiation of echinoid primary mesenchyme cells. Wilhelm Roux’s Arch. 189, 111–122. Harrington, F. E., and Easton, D. P. (1982). A putative precursor to the major yolk protein of the sea urchin. Dev. Biol. 95, 505–508. Hillier, B. J., and Vacquier, V. D. (2003). Amassin, an olfactomedin protein, mediates the massive intercellular adhesion of sea urchin coelomocytes. J. Cell Biol. 160, 597–604. Hirohashi, N., and Lennarz, W. J. (2001). Role of a vitelline layer-associated 350 kDa glycoprotein in controlling species-specific gamete interaction in the sea urchin. Develop. Growth DiVer. 43, 247–255. Hirohashi, N., and Vacquier, V. D. (2002a). High molecular mass egg fucose sulfate polymer is required for opening both calcium channels involved in triggering the sea urchin sperm acrosome reaction. J. Biol. Chem. 277, 1182–1189. Hirohashi, N., and Vacquier, V. D. (2002b). Egg sialoglycans increase intracellular pH and potentiate the acrosome reaction of sea urchin sperm. J. Biol. Chem. 277, 8041–8047. Hirohashi, N., and Vacquier, V. D. (2002c). Egg fucose sulfate polymer, sialoglycan, and speract all trigger the sea urchin sperm acrosome reaction. Biochem. Biophys. Res. Commun. 296, 833–839. Hoshi, M., Nishigaki, T., Kawamura, M., Ikeda, M., Gunaratne, J., Ueno, S., Ogiso, M., Moriyama, H., and Matsumoto, M. (2000). Acrosome reaction in starfish: Signal molecules in the jelly coat and their receptors. Zygote 8(Suppl.), S26–S27.

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Gary M. Wessel and Victor D. Vacquier SeGall, G. K., and Lennarz, W. J. (1979). Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Dev. Biol. 71, 33–48. Selak, M. A., and Sandella, C. J. (1987). Respirational capacity of mitochondria isolated from unfertilized and fertilized sea urchin eggs. Exp. Cell Res. 169, 369–378. Shuy, A. B., RaV, R. A., and Blumenthal, T. (1986). Expression of the vitellogenin gene in female and male sea urchins. PNAS 83, 3865–3869. Silver, R. B. (1986). Isolation of native, membrane-containing mitotic apparatus from sea urchin embryos. Meth. Enzymol. 134, 200–217. Stephens, R. E. (1986). Isolation of embryonic cilia and sperm flagella. Meth. Cell Biol. 27, 217–227. Suzuki, N. (1995). Structure, function and biosynthesis of sperm-activating peptides and fucose sulfate glycoconjugate in the extracellular coat of sea urchin eggs. Zool. Sci. 12, 12–27. Tamura, N., Chrisman, T. D., and Garbers, D. L. (2001). The regulation and physiological roles of the guanylyl cyclase receptors. Endocr. J. 48, 611–634. Unuma, T., Suzuki, T., Kurokawa, T., Yamamoto, T., and Akiyama, T. (1998). A protein identical to the yolk protein is stored in the testis in the male red sea urchin, Pseudocentrotus depressus. Biol. Bull. 194, 92–97. Vacquier, V. D. (1975). The isolation of intact cortical granules from sea urchin eggs: Calcium ions trigger granule discharge. Dev. Biol. 43, 62–74. Vacquier, V. D. (1981). Review: Dynamic changes of the egg cortex. Dev. Biol. 84, 1–26. Vacquier, V. D., and Moy, G. W. (1980). The cytolytic isolation of the cortex of the sea urchin egg. Dev. Biol. 77, 178–190. Vacquier, V. D., and Moy, G. W. (1997). The fucose sulfate polymer of egg jelly binds to sperm REJ1 and is the inducer of the sea urchin sperm acrosome reaction. Dev. Biol. 192, 125–135. Vilela-Silva, A. C., Alves, A. P., Valente, A. P., Vacquier, V. D., and Moura˜ o, P. A. S. (1999). Structure of the sulfated alpha-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: Patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition. Glycobiology 9, 927–933. Vilela-Silva, A. C., Castro, M. O., Valente, A. P., Biermann, C. H., and Moura˜ o, P. A. S. (2002). Sulfated fucans from the egg jellies of the closely related sea urchins Stronyglocentrotus droebachiensis and S. pallidus ensure species-specific fertilization. J. Biol. Chem. 277, 379–387. Walley, T., Terasaki, M., Cho, M.-S., and Vogel, S. S. (1995). Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs. J. Cell Biol. 131, 1183–1192. Ward, G. E., Brokaw, C. J., Garbers, D. L., and Vacquier, V. D. (1985). Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer. J. Cell Biol. 101, 2324–2329. Weidman, P. J., and Kay, E. S. (1986). Egg and embryonic extracellular coats: Isolation and purification. Meth. Cell Biol. 27, 113–137. Wessel, G. M., Marchase, R. M., and McClay, D. R. (1984). Ontogeny of the basal lamina in the sea urchin embryo. Dev. Biol. 103, 235–245. Wessel, G. M., Brooks, J. M., Haley, S., Voronina, E., Wong, J., Zaydfudim, V., and Conner, S. (2001). The biology of cortical granules. Int. Rev. Cytol. 209, 117–206. Wessel, G. M., Berg, L., Adelson, D. L., Cannon, G., and McClay, D. R. (1998). A molecular analysis of hyalin-A substrate for cell adhesion in the hyaline layer of the sea urchin embryo. Dev. Biol. 193, 115–126. Yamaguchi, M., Krita, M., and Suzuki, N. (1989). Induction of the acrosome reaction of Hemicentrotus pulcherrimus spermatozoa by the egg jelly molecules, fucose-rich glycoconjugate and sperm-activating peptide I. Develop. Growth & DiVer. 31, 233–239. Yokota, Y., and Kato, K. H. (1988). Degradation of yolk proteins in sea urchin eggs and embryos. Cell DiVer. 23, 191–200.