JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 17:266-293 (1991)

Multiple Intracellular Signals Coordinate Structural Dynamics in the Sea Urchin Egg Cortex at Fertilization DOUGLAS E. CHANDLER Department of Zoology, Arizona State University, Tempe, Arizona 85287

KEY WORDS

Quick-freezing, Freeze-fracture

ABSTRACT

Fertilization of the sea urchin egg is accompanied by a sequence of structural changes in the egg cortex that include exocytosis, endocytosis, and microvillar growth. This architectural reorganization is coordinated by two intracellular signals: a rapid, transient rise in cytosolic free calcium and a slower, longer lasting increase in cytoplasmic pH. In this report we provide ultrastructural views of these events in quick-frozen eggs and discuss their relationship to the calcium and pH signals.

INTRODUCTION At fertilization the sea urchin embryo receives a full complement of chromosomes by a well-defined sequence of events. The acrosome-reacted sperm binds to the vitelline layer on the surface of the egg, fuses its membrane with the egg plasma membrane, and deposits its chromatin in the egg cytoplasm. A s described in a preceding article (Longo, this volume), sperm chromatin decondenses to form the male pronucleus and joins with the female pronucleus in syngamy. Concurrently, the cortex of the egg undergoes a remarkable structural transformation that includes exocytosis, endocytosis, growth of microvilli, and stiffening of the cortical region through actin polymerization. This transformation results in a n embryo that has a considerably different cell svrface than the quiescent unfertilized egg. The importance of the egg cortex in early development has been outlined in several recent reviews (Longo, 1989; Sardet and Chang, 1987; Schatten, 1982; Schroeder, 1985; Vacquier, 1981) as have the underlying biochemical events in the first few minutes after fertilization (Epel, 1978; Swann et al., 1987; Trimmer and Vacquier, 1986; Turner and Jaffe, 1989; Whitaker and Steinhardt, 1985). Equally striking progress has been made in describing the ultrastructural changes that take place in the egg cortex during this period and it is our goal here to give comprehensive treatment to these studies. In this report we describe the calcium and pH signals that coordinate these ultrastructural changes and we document cortical reorganization at fertilization a s seen in thin sections and freeze-fracture replicas of ultrarapidly frozen eggs. MATERIALS AND METHODS The methods described here are limited to those required for ultrastructural analysis of sea urchin eggs that have been chemically fixed or ultrarapidly frozen within 15 min of fertilization. Lytechinus pictus and Strongylocentrotus purpuratus, obtained commercially (Marinus, Inc., Long Beach, CAI, were kept a t 12°C in aquaria filled with sea water prepared from “Tropic Marin” sea salts (Dr. Biener GMBH, West Germany). Shedding of gametes was induced by injecting 1 ml of

0 1991 WILEY-LISS, INC

0.5 M KC1 into the body cavity, and collecting eggs in sea water. Eggs were dejellied either mechanically by three passages through a 90 pm (L.pictus) or 150 pm ( S .purpuratus) mesh nylon cloth or by suspension in acidified sea water (pH 5.0 for 2 min). The eggs were then washed twice and resuspended in artificial sea water at a cell density of 100 pl packed eggs/ml and maintained at 16°C until use. Sperm was collected “dry” and kept a t 5°C. At 10 min before fertilization, sperm was diluted 1:lOO with sea water, and both sperm and eggs brought to room temperature for all experiments. Fertilization was carried out by mixing 1 ml of a 1% sperm suspension with 6 ml of 3% egg suspension. Then, a t appropriate intervals, eggs were either quick-frozen or fixed by addition of a n equal volume of 4% glutaraldehyde in sea water. Fixation was continued for 1 h at room temperature, and the eggs then washed in sea water. For conventional freeze-fracture, eggs were suspended next in 30%glycerol/70% sea water (viv) for 1.5 h, and packed by centrifugation (1OOg x 1rnin); a drop of packed cells was sandwiched between two gold-alloy specimen carriers and frozen in melting Freon 22. These samples were then fractured in a double replica device in a Balzers 400D freeze-etch unit and replicated with platinum-carbon from a n electron beam gun mounted at a n angle of 45”.Specimens were either fractured a t -130°C without etching or fractured at - 110°C and allowed to etch for 20 s before replication. Replicas were cleaned with bleach, picked up on formvar- and carbon-coated grids, and viewed in a Philips EM 300 microscope a t 80 kV. For conventional thin sectioning, glutaraldehydefixed eggs were postfixed in 1% (w/v) osmium tetroxide in diluted sea water (75% normal tonicity) for 1.5 h a t room temperature, washed in sea water, and blockstained with 1%(w/v) uranyl acetate in 50 mM sodium

Received September 1, 1989; accepted in revised form October 7, 1989. Address reprint requests to Douglas E. Chandler, Department of Zoology, Arizona State University, Tempe, AZ 85287.

SEA URCHIN EGG CORTEX

acetate buffer, pH 5.0, for 1 h in the dark at room temperature. After the cells were dehydrated in a graded series of ethanols and embedded in Araldite 502, silver sections were cut and stained with 10%uranyl acetate in 50% methanol, followed by 0.4% lead citrate in 0.15 M NaOH. Quick-freezing was done as described by Heuser et al. (1979) utilizing a falling plunger to press the sample of eggs against a copper block cooled by liquid helium. A drop of concentrated egg suspension (the pellet from a 1 s, submaximal velocity spin on an Eppendorf microcentrifuge or from 10 to 12 rotations on a hand centrifuge) was placed on a moistened piece of filter paper that was positioned on an aluminum sample disk. A drop of ultrasound conduction gel ('Lectrosonic, Burdick Corp., Minton, WI) was used as a cushion between the sample and the disk. Alternatively, eggs were allowed to settle on polylysine-coated coverglasses, excess eggs washed away, and the eggs fertilized by dipping in a 1%(v/v) sperm suspension. The coverglass, with adherent eggs, was then positioned on a cushion of ultrasound gel or glutaraldehyde-fixed liver sitting on an aluminum disk. The sample was then quick-frozen upon contact with a cold copper block. Quick-frozen specimens to be fractured were clamped down via the aluminum disk to a specially designed specimen table that fit directly on the rotary stage of the Balzers 400D freeze-etch unit. Specimens were fractured superficially with a cold razor blade (so as to expose optimally frozen tissue) at a specimen table temperature of -llo"C, and replicas prepared as described above. Quick-frozenspecimens to be freeze-substituted were placed on frozen acetcae containing 4% osmium tetroxide and covered with liquid nitrogen. The acetone was allowed to thaw and the specimen, now immersed in the acetone-osmium tetroxide mixture, was kept at -70°C for 48 h and then at -20°C for 2 h. The sample was then washed in cold ethanol, block-stained with 1%uranyl acetate in ethanol for 1h in the dark at room temperature, and embedded in Araldite. Specimens t o be deep-etched were fixed in 2% glutaraldehyde in sea water, washed with distilled water four times, and quick-frozen. The frozen specimen was then fractured, etched for 3 min a t a temperature between -100 and -9O"C, and rotary-replicated first with platinum-carbon at an angle of 25" and then with carbon at 75". Isolated egg cortices were prepared on polylysinecoated coverglasses using a glycine-potassium gluconate buffer as described previously (Vacquier, 1975). The cortices were then fixed in 2% glutaraldehyde, passed through distilled water, dehydrated in an ethanol series, transferred to Freon TF, and critical-pointdried using Freon 13. The dried samples were placed on the stage of a Balzers 400D freeze-etch unit at room temperature and rotary-shadowed with platinumlcarbon as described above. Alternatively, critical-pointdried samples were gold-coated in a Hummer I1 sputter coater and viewed in an AMR 1000 scanning electron microscope. Specimens were photographed in stereo using a eu-

267

centric goniometer stage; specimens were routinely tilted 2 6" from horizontal. Freeze-fracture figures are presented as negative images, that is, platinum deposits appear white.

RESULTS AND DISCUSSION The Unfertilized Egg Cortex The cortex of the sea urchin egg is a 2 to 4 km thick region at the egg periphery that consists of the plasma membrane and the organelles and cytoskeletal elements that are linked to this membrane. As seen in thin sections (Fig. 1) the plasma membrane of S. purpuratus eggs exhibits a uniform array of finger-like microvilli. The number and shape of microvilli vary from species to species, being ridge-like in L. pictus and short and stubby in S. purpuratus. The external surface of the cell is covered by a closely adherent vitelline layer that is seen in thin sections as a thin coating of material (arrow, Fig. 1).It is this extracellular coat to which sperm initially bind; its structure is described in detail in Larabell and Chandler (this volume). Just below the plasma membrane is a single layer of cortical granules. Granules in most species are docked in close proximity to the plasma membrane and in S. purpuratus a small plaque of electron dense material can be seen spanning the gap between plasma and granule membrane. The characteristic staining pattern of the granule contents differs for each species. Replicas of eggs that have been quickfrozen in sea water exhibit the same cortex morphology (Fig. 2). Microvilli are arranged in a repeating two-dimensional pattern; cortical granules are often aspherical. Between cortical granules is an extensive network of endoplasmic reticulum. This network takes on the appearance of vesicles in thin sections (Fig. 1)but its true extent is best seen in intact eggs that have been quickfrozen and deep-etched (Figs. 3 and 4). Stereo pairs reveal complex strands of organelles that are anchored to the inside surface of the plasma membrane and extend into the cytoplasm (arrows, Fig. 3). These strands are heavily decorated with clusters of small particles. Similar networks can be seen several micrometers into the cortex (Fig. 4). In some cases, the fracturing process has split the endoplasmic reticulum (ER) to reveal the E face (arrows) and P face (asterisk) of underlying tubular and vesicular membranes. The cortex is a structurally integrated component of the egg and can be isolated from the remainder of the egg by several experimental techniques. First, early observations indicated that sea urchin eggs can be stratified by centrifugation, typically by use of 9,OOOg forces for 15 min (Anderson, 1970). The egg interior becomes layered into regions containing lipid droplets, pronucleus, Golgi and ER, mitochondria, yolk granules, and pigment granules, as one proceeds from the centripetal to the centrifugal pole. The cortical granules, however, remain firmly attached to the egg plasma membrane and these structures with associated cortical ER and cytoskeletal components constitute a structurally identifiable cortex. More recently, another method to isolate the egg cor-

268

D.E. CHANDLER

Fig. 1. Cortex of an unfertilized S. purpurutus egg showing vitelline layer (arrow), cortical endoplasmic reticulum, and cortical granules. Specimen was quick-frozen and freeze-substituted. x 51.000.

Fig. 2. Freeze-fracture replica of the unfertilized egg cortex. The plasma membrane displays a regular array of microvilli and just below is a single layer of cortical granules. x 34,000.

tex has used homogenization and differential centrifugation (Detering et al., 1977; Haggerty and Jackson, 1983;Mabuchi and Sakai, 1972; Sasaki and Epel, 1983; Vacquier and Moy, 1980) or sticking the egg surface to polylysine-coated coverglasses and shearing away the remainder of the cell using a stream of buffer Wac-

quier, 1975,1976). Both techniques result in isolation of the egg cortex as a cell fragment that can be analyzed either biochemically or ultrastructurally. Isolated cortices prepared by shearing consist of discrete patches of plasma membrane, curled at the edges, that are covered on their interior surface with a uniform

SEA URCHIN EGG CORTEX

269

Fig. 3. Strands of endoplasmic reticulum extend into the cytoplasm from their attachment sites at the plasma membrane (arrowheads). Specimen was fixed, quick-frozen, deep-etched, and rotaryshadowed. Stereo pair. x 65,000.

Fig. 4. Endoplasmic reticulum deeper in the cortex. These chains of vesicles are decorated with particles and exhibit E and P fracture faces (arrowheads and asterisks, respectively). Sample prepared as in Fig. 3. Stereo pair. x 65,000.

array of cortical granules (Fig. 5). In platinum replicas, cortical granules are linked to each other, and to the plasma membrane below by 6 nm diameter filaments

(Fig. 6). Also attached to the inner surface of the plasma membrane is a n extensive array of cortical ER tubules that are joined by single filaments into polyg-

270

D.E. CHANDLER

Fig. 5. Isolated cortices from S.purpurutus eggs as seen by SEM. x 1000.

Fig. 6. Cortical granules adhere to the isolated cortex and are interconnected by 6 nm filaments. Specimen was critical-point-dried and rotary-shadowed. x 70,000.

SEA URCHIN EGG CORTEX

271

Fig. 7. Organelles seen on the inner aspect of the cortex include cortical granules (cg), small granules (s),and polygonal arrays of endoplasmic reticulum (er). Specimen prepared as in Fig. 6. Stereo pair. x 27,000.

Fig. 8. Chains of ER vesicles are in close contact with the plasma membrane. Specimen prepared as in Fig. 6. Stereo pair. X 66,000.

onal arrays (Fig. 7; see also Chandler, 1984c; Sardet, 1984).At higher magnification, these tubules appear to be decorated with particles (Fig. 8) that are thought to be ribosomes, based on their sensitivity to RNAase (Sardet, 1984). This cortical ER network is thought to

be the organelle that releases calcium at fertilization to trigger cortical granule exocytosis. Indeed, Poenie and Epel(l987)have used cytochemical techniques to show that these organelles contain large amounts of calcium. In addition, Henson et al. (1989) have shown the pres-

272

D.E. CHANDLER

Fig. 9. Microvilli in the unfertilized egg exhibit a microfilament network within their interiors. Specimen was quick-frozen and freezesubstituted. x 96,000. Fig. 10. Fluorescence microscopy of the unfertilized egg surface demonstrates a punctate localization of fluorescein-conjugated phal-

ence of a calsequestrin-like protein in this network, suggesting that its function may parallel that of sarcoplasmic reticulum in muscle. In whole eggs, the cortex has been shown to contain large amounts of G-actin (Bonder et al., 1989; Spudich and Spudich, 1979; Vacquier and Moy, 1980; Wang and Taylor, 1979), and more recently the presence of F-

lacidin similar to the pattern seen for microvilli. (C. Merkle, unpublished observations). x 5,000. Fig. 11. Microvilli, viewed from inside the egg cortex, have filaments extending from their interior. X 94,000.

actin has been found in the numerous microvilli that dot the egg surface (Hensen and Begg, 1988). In thin sections of eggs that have been quick-frozen and freezesubstituted one can see prominent filaments that course through the shaft of each microvillus (Fig. 9). When whole eggs are stained with fluorescein-conjugated phallacidin, which specifically binds to F-actin,

SEA URCHIN EGG CORTEX

one can see a punctate pattern of fluorescence (Fig. 10) that exactly matches the distribution of microvilli (Hensen and Begg, 1988). Furthermore, if isolated cortices are either critical-point-dried or quick-frozen and freeze-dried one can detect a tuft of filaments that extend from the base of each microvillus (Fig. 11)that in other studies have been shown to label with the S-1 fragment of myosin (Hensen and Begg, 1988). Thus, the unfertilized egg cortex contains large amounts of actin, most of which is soluble G-actin that waits in storage for the rapid assembly of microfilaments and growth of microvilli just after fertilization.

273

Kidd, 1978; Longo et al., 1986; Rossignol et al., 1984). Subsequent fusion of the two gametes occurs first at the acrosomal process of the sperm, but soon the whole cell is engulfed by egg cytoplasm that wells up around the sperm-the so-called fertilization cone (Schatten and Schatten, 1980; Tilney and Jaffe, 1980). The sperm, now inside the egg, rotates 180“, its nucleus begins to decondense, and an aster is formed that over a period of minutes will help propel the newly formed male pronucleus toward the center of the egg where it will undergo syngamy with the female pronucleus (Schatten, 1982; Schatten, 1981; Longo, this volume). Even before pronuclear migration, however, sperm fusion with the egg initiates a striking series of events Intracellular Signals at Fertilization in the egg cortex-the “cortical reaction.” Starting at Fertilization consists of a complex series of events the point of sperm entry, cortical granules fuse with that is initiated by sperm-egg fusion, and is sequen- the plasma membrane. This exocytotic event begins tially followed by architectural reorganization of the about 20 s after insemination and during the next 30 s egg cortex, metabolic activation, and finally joining of spreads as a wave over the egg surface to the opposite the male and female pronucleus to form the first zygote pole (Anderson, 1968; Chandler and Heuser, 1979; nucleus (Fig. 12). Sperm binding to the vitelline layer Eddy and Shapiro, 1976; Schuel, 1985). Exocytosis on the surface of the egg occurs within seconds (Chand- leads to elevation of the vitelline layer and provides ler and Heuser, 1980a; Glabe and Vacquier, 1978; enzymes and structural proteins that, within 5 min

D.E. CHANDLER

274

after insemination, transform this layer into the chemically and mechanically tough fertilization envelope (Kay and Shapiro, 1985; Larabell and Chandler, this volume). Exocytosis is soon followed by what is likely to be a compensatory burst of endocytosis that rapidly removes membrane from the egg surface between 2 and 4 min after insemination (Chandler and Heuser, 1979; Fisher and Rebhun, 1983; Schroeder, 1979). During the same period actin polymerization in the cortex leads to an experimentally measurable increase in cell surface stiffness (Hiramoto, 1974; Mitchison and Swann, 1955) and to growth of long finger-like microvilli (Chandler and Heuser, 1981; Eddy and Shapiro, 1976; Fisher and Rehbun, 1983). Finally, during the first 15 min after sperm-egg fusion, the egg is changed from a metabolically dormant cell to one that is actively synthesizing new protein and

1.31N DAG

1.o

CA*+

WAVE

Fig. 13. Time course of intracellular signals at fertilization. Top panel: The membrane potential abruptly depolarizes to + 20 mV and slowly repolarizes over several minutes. Second and third panels:A transient increase of both inositol-1,4,5-triphosphate (IP,) and diacyl-

6.8

r~ 0

I

I

1

I

A

I

2 TIME (MIN.)

I

1

3

.

5

glycerol (DAG) in the first minutes is followed by a longer lasting increase at 2 min and beyond. Fourth panel: The calcium wave, as judged by the percentage of cells undergoing cortical granule exocytosis, occurs during the initial IP, increase but exhibits a lag of about 10 to 15 s. Bottom panel: Cytoplasmic alkalinization occurs between 1and 3 min after fertilization. (The first panel is redrawn from Chambers and de Armendi, 1979, with permission of Academic Press; the second, third, and fourth are redrawn from Ciapa and Whitaker, 1986, with permission of Elsevier Science Publishers BV; the last is redrawn from Shen and Steinhardt, 1978,with permission of Macmillan Magazines Ltd.)

Fig. 14. Scheme depicting hydrolysis of phosphatidylinositol-4,5-bisphosphate(PIP,) to inositol-1,4,5trisphosphate (IPJ and diacylglycerol (DAG) in the sea urchin egg plasma membrane at fertilization. R, putative sperm receptor; G , putative GTP-binding protein; PL, inositol-specific phospholipase C; kinase C, protein kinase C; CG, cortical granule.

SEA URCHIN EGG CORTEX

275

SPERM-EGG FUSION

GTP BINDING PROTEIN

PHOSPHOLIPASE C PIP,I

Cortical Reticulum

t

Cytoplasmic ~ a 2 + 4

t

Cortical ‘Granule Exocrosis

t

Elevation of Vitelline Layer

t

Assembly of Fertilization Envelope

*IPS-

+ DAG

Activation of Protein Kinase C

J

t

Protein Phosphorylation

t?

Na+ / H+ Exchange

t

\4Cytopiahc PH

/

J

Metabolic Activation

\

4

\

Actin Polymerization Microvillar Growth

and (IP,) diacylglycFig. 15. Actions of inositol-l,4,5-triphosphate erol (DAG) at fertilization in the sea urchin egg. IP, induces release of calcium from the cortical endoplasmic reticulum, which in turn induces exocytosis; this action can be mimicked by calcium ionophores such as A23187. DAG activates protein kinase C, which in turn may

phosphorylate the Na’lH’ exchanger. Phorbol esters such as phorbol myristate acetate (PMA) can mimic DAG while salts of weak bases such as ammonia can induce cytoplasmic alkalinization as does the Na’lH’ exchanger. Steps marked are speculative a t present. PIP,, phosphatidylinositol-4,5-biphosphate.

nucleic acids in preparation for the first cell division occurring 90 min later. Increased synthesis of NADP and its conversion to NADPH provides a stockpile of reducing equivalents for anabolic reactions and for production of hydrogen peroxide (Epel, 1964; Epel et al., 1981; Whitaker and Steinhardt, 1981). Reducing equivalent production as well as ATP production requires a vastly increased oxygen consumption that is seen first as a burst between 1 and 3 min after insemination but that remains a t a greater than basal level thereafter (Foerder et al., 1978; Steinhardt and Epel, 1974; Steinhardt et al., 1972). The rate of protein synthesis increases over 20-fold within 15 min of insemination and remains high throughout early development (Dube et al., 1983; Grainger et al., 1979; Winkler et al., 1980). DNA synthesis begins just after syngamy and heralds the start of the S phase (Dube et al., 1983; Hinegardner, 1964). This series of events at fertilization is coordinated by three intracellular signals: plasma membrane depolarization, a transient rise in the cytosolic free calcium level, and a relatively permanent increase in cytosolic pH. Membrane depolarization from -70 mV to +20 mV occurs within seconds of sperm-egg fusion as a result of a calcium-dependent action potential followed by an increased cation conductance (Fig. 13, top panel) (Chambers and de Armendi, 1979; David et al., 1988;

Jaffe, 1976; Jaffe and Robinson, 1978). Voltage clamp studies have shown that it is actually preceded by a transient activation current that represents the first experimental sign of sperm-egg fusion (Longo et al., 1986; Lynn et al., 1988).These studies have also shown that depolarization of the plasma membrane prevents other sperm from fusing and thus represents what is called the “fast block” to polyspermy (Jaffe, 1976,1986; Jaffe et al., 1982; Lynn and Chambers, 1984; Shen and Steinhardt, 1984; Whitaker and Steinhardt, 1983). The calcium and pH signals that follow appear to originate from the phosphatidylinositol cycle utilized in many cell responses that involve intracellular calcium signals (Berridge, 1987; Majerus et al., 1984; Turner and Jaffe, 1989). Central to the pathway (as shown in Fig. 14) is the receptor/GTP-binding protein regulation of an inositol-specific phospholipase C that hydrolyzes phosphatidyl-4,Ei-biphosphate (PIP,) to inositol-1,4,5-triphosphate(IP,) and diacylglycerol (DAG) (Gilman, 1987; Gomperts et al., 1986). The IP, produced enters the cytoplasm, and in many cells is known to induce release of calcium sequestered in the ER (Berridge, 1987). DAG, being lipid-soluble, remains in the plasma membrane and there activates protein kinase C, which in turn phosphorylates various effector proteins (Kikkawa and Nishizuka, 1986; Nishizuka, 1984,1988).

‘I?”

D.E. CHANDLER

276

‘“I

F l

r

*’O0

I

3 ’Yo

20

+

1

30

40

+

TMB-8

50

60

70

TIME (Minutes) Fig. 16. 45Ca”+ efflux from sea urchin eggs at fertilization. L . pzctus eggs were preloaded with 45Ca2+and washed, and efflux of tracer was measured. Eggs were fertilized a t the arrow ( 0 , A) or remained unfertilized. TMB-8,100 pM, was present between the arrows (A, A) and completely inhibited the fertilization-induced release of calcium. (Reproduced from Stapleton et al., 1985.)

In sea urchin eggs fertilization is accompanied by a n increased turnover of phosphatidylinositol, a measurable decrease in PIP, (Turner e t al., 1984), and a n immediate but transient increase in both IP, and DAG, a s shown in Fig. 13, 2nd and 3rd panels (Ciapa and Whitaker, 1986; Kame1 et al., 1985; Whitaker and Aitchison, 1985). Hydrolysis of PIP, to form IP, and DAG may be initiated by activation of a GTP-binding protein as in other cells (Jaffe et al., 1988; Turner et al., 1986; Turner and Jaffe, 1989) based on the fact that nonhydrolyzable analogues of GTP (e.g., GTP-y-S) will activate the signaling cascade (Turner et al., 1986). However, many investigators are currently questioning the involvement of a GTP-binding protein in this cascade since certain observations have contradicated this hypothesis. Specifically, GTP-y-S, which should inhibit GTP-binding protein activation of the phospholipase C, and therefore the IP,-induced calcium wave, does not do so (Whitaker et al., 1989). Clearly, more work must be done to prove or disprove the existence of a GTP-binding protein and a putative “sperm fusion” receptor that activates the GTP-binding protein. Regardless of this controversy, i t is now firmly established that one of the messengers produced in eggs, IP,, will lead to calcium release, a rise in intracellular free calcium, cortical granule eXOCytOSiS, and finally elevation and assembly of the fertilization envelope , either isolated egg tor( ~ i15). ~ .Application of ~ p to tices Or to purified ER from eggs induces calcium release as measured by minielectrodes or by

~ i17.~ The . calcium wave in an L, egg as visualized by the calcium sensitive fluorescent probe Fura-2. A The wave front is seen in white at 7 o’clock superimposed on lower basal calcium levels shown in grays and black. B and C: The wave front sweeps across the egg to the opposite pole leaving high (white) calcium levels. (Reproduced from Tsien and Poenie, 1986, with permission of Elsevier Science Publishers BV.)

SEA URCHIN EGG CORTEX

*I-

-

277

r 2.0

v)

z W Iz -

1.O

W

0

0.3

z W

0 v)

w a 0

3 lL

L

U 2 MIN.

Fig. 18. Cytoplasmic alkalinization in S . purpurutus eggs in response to phorbol ester (PMA) as monitored by the pH-sensitive fluorescent probe BCECF. The increase in cytoplasmic fluorescence (pH) between 30 s and 3 min after PMA addition is dose-dependent from 0.3 to 2.0 )LMPMA. (N. Mozingo, unpublished observations).

45Ca2+movements (Clapper and Lee, 1985; Oberdorf et al., 1986). Furthermore, injection of IP, into eggs initiates cortical granule exocytosis (Whitaker and Irvine, 1984), and this reponse is blocked if calcium levels within the cell are buffered by EGTA (Turner et al., 1986). Release of internal calcium stores seems t o account fully for the calcium signal in sea urchin eggs since removal of extracellular calcium is without effect (Crossley et al., 1988; Schmidt et al., 1982). Release of sequestered calcium just after fertilization can be measured in one of three ways. First, as shown in Fig. 16 (circles), eggs preloaded with 45Ca2+exhibit a rapid efflux of tracer calcium upon insemination (Stapleton et al., 1985; Steinhardt and Epel, 1974). This efflux is blocked if TMB-8, an inhibitor of ER calcium release, is present (solid triangles, Fig. 16). Second, calcium release can be measured either by the calcium-sensitive photoprotein aequorin or by fluorescence probes such as fura-2 as illustrated in Fig. 17 (Eisen et al., 1984; Steinhardt et al., 1977; Swann and Whitaker, 1986; Tsien and Poenie, 1986). In Fig. 17a, release begins at about 7 o’clock and is seen as a white wave of high calcium levels superimposed on lower basal calcium levels shown in grays and black. This wave sweeps across the face of the egg in Fig. 17b and finally completes its transit in Fig. 17c, in which the whole egg cytoplasm now shows high (white) calcium levels. This calcium wave, about 2 pM at its peak (Tsien and Poenie, 19861, is sufficient to trigger exocytosis. Isolated egg cortices, for example, undergo exocytosis in vitro, the response being half maximal at

about 1 pM (Jackson et al., 1985; Jackson and Crabb, 1988; Moy et al., 1983; Whitaker and Baker, 1983). DAG, the other product of PIP, hydrolysis, may be an equally important signal in egg activation. DAG and phorbol myristate acetate (PMA),activators of protein kinase C, both cause an alkalinization of the egg cytoplasm that can be detected both by pH-sensitive microelectrodes (Shen and Burgart, 1986) and by fluorescence probes such as dicarboxyfluorescein (Swann and Whitaker, 1985) and bis(carboxyethy1)carboxyfluorescein (BCECF; see Fig. 18). This pH change is similar to that seen just after fertilization (Fig. 13, bottom panel), wherein the cytosolic pH increases from 6.8 to 7.2 during the period from 1 to 3 min after insemination (Dube et al., 1985; Epel et al., 1974; Grainger et al., 1979; Shen and Steinhardt, 1978, 1979). This pH rise is known to result from activation of an amiloridesensitive Na+/H+ exchanger in the egg plasma membrane (Johnson and Epel, 1981; Johnson et al., 1976; Shen and Steinhardt, 1978,1979).Increased H+ efflux and Na+ influx are seen 2 to 4 min postinsemination as measured by ion-sensitive microelectrodes (Shen and Burgart, 1985; Shen and Steinhardt, 1979). As indicated in Fig. 15, cytoplasmic alkalinization seen at fertilization initiates a dramatic increase in protein synthesis and is also required for processes such as karyokinesis, microvillar growth, chromosome decondensation, and ultimately DNA synthesis and mitosis (Dube et al., 1985). Experimental alkalinization of the egg cytoplasm by ammonia will initiate many of these events in the absence of calcium-initiated events such

278

D.E. CHANDLER

A ' B "C4D

as cortical granule exocytosis (Epel et al., 1974; Mazia, 1974). Indeed, the pH increase induced by PMA has also been shown to increase protein synthesis (Swann and Whitaker, 1985). What is not known is the molecular mechanism that activates the N a + / H + exchanger. There is growing evidence that protein phosphorylation can regulate ion channels and transport proteins including the Na + / H f exchanger (Grinstein and Rothstein, 1986; Levitan, 1985). One possibility, still speculative, is that protein kinase C activation a t fertilization leads to phosphorylation of either the N a + / H + exchanger or a protein that regulates the exchanger. Recently, PMA was shown to induce increased phosphorylation in over two dozen egg proteins (Chandler and Vacquier, 1988). If isolated cortices are prepared from such eggs one finds that three of these proteins with molecular masses of 240, 92, and 80 kD are associated with the egg cortex (lane B', Fig. 19). Further fractionation of the cortex into isolated cortical granules (lane C', Fig. 19) and a plasma membraneivitelline layer complex (PMVL; lane D', Fig. 19) shows that these phosphorylated proteins remain with the PMVL complex, a finding consistent with modification of a plasma membrane protein like the N a + / H + exchanger. However, not all evidence agrees with such a hypothesis. Shen (1989a,b) has recently shown that inhibitors of PKC, such as K252a and H7, will block pH increases elicited by dioctanoylglercerol and PMA, respectively, but do not block the pH rise associated with fertilization. In contrast, W-7, a n inhibitor of Ca2 -calmodulin-activated enzymes, does inhibit the fertilization-induced pH rise. Clearly, more work needs to be done to identify the protein kinase that phosphorylates and thereby regulates the Na + / H f exchanger.

Fig. 19. Phosphorylation of the 92 kD and 80 kD bands in egg cortices isolated from eggs treated with PMA. Eggs were labeled with 32po 3 - and treated for 10 min with 1 pM PMA, and proteins were analyzed by SDS-PAGE. Left: Silver-stained gel. Right: Corresponding autoradiogram. Samples were as follows: A: Whole eggs. B: Cortical lawns. C: Isolated cortical granules. D Cortical lawns with granules removed (PMVL complex). Bands at 92 kD and 90 kD appear enriched in the PMVL complex (lane D') but are absent in isolated cortical granules (lane C'). (Reproduced from Chandler and Vacquier, 1988, with permission of the publisher.)

Cortical Granule Exocytosis The wave of cortical granule exocytosis that sweeps across the egg surface can be seen a t both the light microscopic and electron microscopic levels. In thick sections one can see a transient concavity in the egg at the point where the wave begins in the 2 o'clock position (Fig. 20a); small exocytotic pockets can be seen a t the front of the wave as it spreads toward the opposite pole (arrows, Fig. 20a). The wave is more easily seen as a two-dimensional array of exocytotic pockets in freezefracture replicas of the egg surface. In Fig. 20b, we have photographed such a replica and blackened those areas that represent fused granule membrane. Again it is clear that the wave is progressing from 2 o'clock toward the 8 o'clock position. At the front of the wave single exocytotic pockets are few and far between, while in the middle of the wave they have become abundant enough to lie adjacent to each other i n many cases. Finally, a t the rear of the wave, membranes from fused granules have involved the whole area and have become confluent. The wave progresses over the egg surface at a velocity of about 5 pmls. The mechanism of propagation is not known but there is at least one hypothesis suggested by Whitaker (Swann and Whitaker, 1986) that is illustrated in Fig. 21 and is based on properties of the phosphatidylinositol signaling system. The key asser-

tion is that the inositol-specific phospholipase that produces IP, from PIP, is calcium-activated over the range of calcium concentrations seen during the calcium wave. Indeed, a phospholipase C dependence on calcium has been observed in hepatocytes (Uhing et al., 1986) and in sea urchin eggs (Whitaker and Aitchison, 1985); in addition, the kinases that are thought to synthesize PIP2 in sea urchin eggs are calcium-dependent (Oberdorf et al., 1989). What might happen is that calcium is released first at the point of sperm entry, either from the sperm directly or more likely from sperm fusion-mediated PIP, hydrolysis. This, in turn, produces IP,, which initiates calcium release from the cortical ER. The calcium released not only triggers exocytosis locally but also diffuses through the cortex ahead of the secretory wave to activate inositol-specific phospholipase C and produce additional IP,. Thus, the wave would be propagated by a positive feedback cycle of calcium release generating a spreading wave of IP, production, which, in turn, results in further spread of calcium release. Certain features of this model agree with what we know about the calcium wave in many eggs including the Medaka (Gilkey, 1983; Gilkey e t al., 1978; Jaffe, 1983). In this fish egg, propagation of the calcium wave can be "rerouted" around a n area that

+

A BCD 205 116 97 66

45

21 14

SEA URCHIN EGG CORTEX

Fig. 20. The wave of cortical granule exocytosis as seen by light microscopy (A) and in freeze-fracture replicas (B). A The wave was initiated at 2 o’clock and has propagated around the sides. At the wave front there are numerous small elevations of the vitelline layer (arrowheads) that correspond to sites where single granules have

279

been released. x 900. B: Sites where granule membranes have fused with the plasma membrane are blackened out t o illustrate the gradient of exocytosis as the wave passes over the “face” of the egg. x 2,000. (Reproduced from Chandler and Heuser, 1979, with permission of the Rockefeller University Press.)

DIRECTION OF PROPAGATION Fig, 21. Hypothetical model of exocytotic wave propagation in the sea urchin egg a t fertilization. Activated inositol-specific phospholipase C (PL*) produces inositol-1,4,5-triphosphate (IP,), which in turn elicits calcium release from the cortical endoplasmic reticulum (small

circles). The calcium released spreads out to activate phospholipase C (PL) ahead of the exocytotic wave. This activation leads to further IP, production and calcium release in a positive feedback cycle.

has been microinjected with EGTA to buffer the calcium rise. In addition, propagated waves can be initiated in the Medaka by localized application of calcium ionophores such as A23187 (Gilkey et al., 1978) although, surprisingly, this does not appear to be the

case for the sea urchin egg (Chambers and Hinckley, 1979). One might expect that the wave-like nature of exocytosis in sea urchin eggs would make it an excellent model system in which to study the steps in fusion of

280

D.E. CHANDLER

Fig. 22. Cortical granules are docked at the plasma membrane at sites containing a small patch of electron dense material (arrow). Specimen was quick-frozen and freeze-substituted, X 192,000. Fig. 23. Freeze-fracture replica of a quick-frozen egg shows that cortical granule exocytosis begins with a small, 22 nm diameter pore (arrow) that has been stabilized by hyperosmotic, stachyose-con-

taining sea water. (Reproduced from Chandler et al., 1989, with permission of the Rockefeller University Press.) Fig. 24. As the pore widens, secretory proteins decondense and are forced outward into the extracellular space. Specimen was quickfrozen and freeze-substituted. x 62,000.

281

SEA URCHIN EGG CORTEX

0

0

0

MEMBRANE ADHESION

FUSION I PORE FORMATION

PORE WIDENING

@ GRANULE-

I

: HYPEROSMOLALITY

POLYMERS; DIVALENT CATIONS

Fig. 25. Sequential steps in cortical granule exocytosis. Reproduced from Chandler et al., 1989, with permission of the Rockefeller University Press.

secretory granules with the plasma membrane. Early stages of membrane fusion should predominate a t the front of the wave while later stages would be seen at the middle or rear of the wave. In actual practice, individual fusion events are much faster than propagation of the wave, and these events are nearly complete even a t the wave front (Chandler and Heuser, 1979). Thus, in order to visualize individual steps in the membrane fusion we have employed quick-freezing to stop fusion within a few milliseconds and hyperosmotic media to stabilize early steps in the process (Chandler 198413; Chandler et al., 1989). Before fusion, each cortical granule is docked at the plasma membrane. The space between the granule and plasma membrane, at their closest approach, is filled with a dense osmiophilic material (arrow, Fig. 22). What this material is and whether it plays a role in initiating membrane fusion is not known. Fusion starts with formation of a single small pore 20 to 40 nm in diameter (Fig. 23) that continues to grow in size as granule contents decondense and are discharged into the sea water (Fig. 24). The smallest pores (e.g., Fig. 23) are seen only when eggs are exposed to hyperosmotic sea water containing low molecular weight osmoticants such as sucrose, sodium sulfate, and stachyose (Chandler et al., 1989; Merkle and Chandler, 1989). Hyperosmotic media induce polymerization of actin in the sea urchin cortex resulting in formation of a granule-free zone that pushes most granules away from the plasma membrane, thus preventing their fusion (Merkle and Chandler, 1989). The minority (about 10%) that remain docked, however, undergo fusion, which is abruptly halted after pore formation. Our current thought is that widening of the pore is prevented either because the cortex has been stiffened by actin polymerization, thus limiting granule swelling, or, alternatively, that

hyperosmolality blocks the conformational changes that secretory proteins undergo during granule matrix decondensation. High molecular weight polymers also block granule discharge but a t a later stage and with very little increase in osmolality. Incubation of eggs in 10 kD dextran (T10, Pharmacia; average molecular weight) followed by activation of exocytosis with the calcium ionophore A23187 results in complete opening of exocytotic pockets. However, granule cores do not dissociate and the pockets surrounding them do not become integrated with the cell surface. These data substantiate the conclusion reached by Whitaker and Zimmerberg (1987) that polymer solutions block exocytosis after membrane fusion. The fact that dextran solutions isosmotic with sea water also block granule matrix dispersal indicates that total osmotic strength itself is not the determining factor in inhibition. Rather, this inhibition depends strongly on the molecular weight of the polymer; Whitaker and Zimmerberg (1987) have shown at the light microscopic level that polymers above 3,500 daltons inhibit matrix dispersal while those below this figure do not unless very high osmolalities are reached. This finding suggests that high molecular weight polymers are unable to enter the granule matrix and therefore reduce water activity outside the matrix and retard water flow into the granule core during disassembly. Inhibition of exocytosis by low molecular weight OSmoticants and by high molecular weight dextrans at specific stages allows one to separate the complete process into four steps (Fig. 25): membrane adherence, membrane fusionlpore formation, pore widening, and granule matrix discharge. Membrane adherence is characterized by dimpling of the plasma membrane inward and contact of the two membranes in a highly localized region (Chandler et al., 1989; Zimmerberg et

282

D.E. CHANDLER

Fig. 26. Exocytosis is followed by a burst of endocytosis between 2 and 4 min after fertilization. Several coated pits (arrows) can be seen in this S. purpurutus egg fixed at 2 min postinsemination. (Reproduced from Chandler and Heuser, 1979, with permission of the Rockefeller University Press.) x 40,000.

Fig. 27. Freeze-fracture replica of the egg surface 2 min after insemination. Coated pits are interspersed among the stump-like bases of microvilli. Specimen was fixed, glycerinated, and frozen in freon. (Reproduced from Chandler and Heuser, 1979, with permission of the Rockefeller University Press.) x 25,000.

SEA URCHIN EGG CORTEX

Fig. 28. Sequential steps in endocytosis as seen in replicas of the plasma membrane P face in quick-frozen S . purpuratus eggs. A Presumptive sites of endocytosis are marked by gatherings of large IMPs (arrows). ELD The membrane dips inward (B), the IMPs are pulled together (C), and finally a narrow neck forms having a tiny opening

283

to the cell surface (D); large IMPs are no longer visible. E: The neck can be quite long and is bare; the vesicle a t the bottom of the pit is covered with a clathrin basket. Isolated cortex from a fertilized egg that has been critical-point-dried and rotary-shadowed. A-D, x 150,000; E, x 294,000.

284

D.E. CHANDLER

Fig. 29. Aerial view of the egg surface 2 min after insemination showing the “forest” of microvilli that have grown. Specimens in Figs. 28-32 were fixed, quick-frozen, deep-etched, and rotary-shadowed. Stereo pair. x 5,300.

al., 1985). Dimpling of the plasma membrane is similar croanalysis has shown that calcium is lost rapidly from to that described in mast cells (Chandler and Heuser, the cyst matrix during release (Lubbock et al., 1981). 1980b), and amebocytes (Ornberg and Reese, 1981). Step two, membrane fusion, occurs within the region of Endocytosis adherence through formation of a small pore. UltraExocytosis in sea urchin eggs, as in many secretory structural studies in amebocytes (Ornberg and Reese, 19811, mast cells (Chandler and Heuser, 1980b; Chand- cells, is quickly followed by endocytosis. Between 2 and ler, 1984a), and sea urchin eggs (Chandler, 1984b) in- 4 min after insemination numerous coated pits can be dicate the presence of pores 10 to 30 nm in diameter, seen at the cell surface (arrows, Fig. 26). In freeze fracwhile electrophysiological studies in mast cells (Zim- ture replicas the P face of the plasma membrane is merberg et al., 1987) suggest that initial pores may be studded with the stump-like bases of crossfractured mia s small as 1or 2 nm in diameter. Clearly, pore forma- crovilli (Fig. 27). Between microvilli the plasma memtion requires membrane adherence since separation of brane is covered with endocytic figures a t every stage cortical granules from the plasma membrane in hyper- of invagination (Fig. 27). In quick-frozen cells, these osmotic media (> 2.0 osmolikg) blocks granule fusion images can be placed in a sequence that suggests the completely. The third step, pore widening, is blocked in consecutive events in the formation of a n endocytic veshyperosmotic media. Pore widening is also slowed by icle (Fig. 28). Presumptive sites of endocytosis consist hyperosmotic solutions in beige mouse mast cells (Zim- of gatherings of large intramembrane particles (IMPS) merberg et al., 1987). In this system, electrophysiolog- on the plasma membrane P face (arrows, Fig. 28a). The ical measurements show that pore widening is a n ex- plasma membrane bows inward (Fig. 28b), producing a tremely variable and dynamic process (Curran et al., well-defined depression in the cell surface (Fig. 28c). 1988). Further experiments are needed to discern The vesicle formed is attached to the cell surface by a which cellular elements are responding to osmotic narrow neck until it is finally pinched off (Fig. 28d,e). In both thin sections (Fisher and Rebhun, 1983) and in pressure a t this step. In the fourth and final step, the granule matrix must platinum replicas of egg cortices prepared from fertilbe discharged into the extracellular space. This step ized eggs (Fig. 28e) these necks can appear quite long appears to require water movement into the matrix and free of clathrin while the vesicle a t the tip of the since it is inhibited by polymers that, due to their ex- neck is covered by the usual clathrin lattice. The exact events that initiate and control endocytoclusion, selectively reduce water activity outside of the matrix. Dispersal is also retarded by the presence of sis are not known. Since endocytosis, like other events divalent cations (Whitaker and Zimmerberg, 1987); in cortex reorganizaton, can be triggered by application one possibility is that calcium and/or magnesium ions of the calcium ionophore A23187, i t seems likely that help bind the matrix together by charge interaction the calcium signal is involved. Exocytosis does not need with anionic matrix constituents and that calcium to precede endocytosis. If high pressure (7,000 psi) is must be displaced during matrix discharge. A similar used to block cortical granule exocytosis just after fersequence may occur in nematocyst discharge: x-ray mi- tilization, endocytosis and microvillar growth ensue,

SEA URCHIN EGG CORTEX

Fig. 30. Microvilli on the egg surface at 30 s postinsemination are flaccid and exhibit numerous varicosities along their length. Stereo pair. x 14,000.

285

Fig. 31. Microvilli at 2 min postinsemination appear finger-like and extend from broad pedestal-like bases on the cell surface. Stereo pair. x 36,000.

286

D.E. CHANDLER

Fig. 32. At 5 rnin postinsemination microvilli appear erect and extend from thin cytoplasmic sheets. Stereo pair. x 48,000.

although their timecourse is somewhat slower (Fisher et al., 1985).

Microvillar Growth Between 1 and 5 min after fertilization there is a rapid growth of microvilli on the egg surface (Chandler and Heuser, 1981). Secondary elongation of these microvilli occurs at approximately 16 rnin after insemination (Schroeder, 1978). We will limit ourselves here to the initial growth phase, which we have visualized in stereo pairs of quick-frozen, deep-etched specimens. Overall, the newly fertilized egg will develop a thick

forest of erect, multifingered microvilli (Fig. 29). Growth begins with formation of single, thin microvilli having multiple varicosities along their length (Fig. 30). Such growth begins on cell surface areas adjacent to exocytotic pockets even before the exocytotic wave has passed (Chandler, unpublished observations). The beaded appearance of these microvilli disappears within 1.5 rnin after insemination and by 2 min groups of finger-like microvilli have been lifted off the egg surface on broad-based pedestals (Fig. 31). Within 5 min after insemination, the fingers have become long, thin spindles that insert into raised sheets of cytoplasm (Fig. 32). At all stages, the surfaces of these microvilli

SEA URCHIN EGG CORTEX

287

Fig. 33. At 2 min postinsemination microvilli have not grown in eggs treated with cytochalasin B. X 53,000.

Fig. 34. Thin sections of fertilized S. purpurutus eggs exhibit broad-based microvilli and a cortex filled with cytoskeletal filaments but relatively few organelles. Specimen was quick-frozen 4 min postinsemination and freeze-substituted. x 50,000.

are covered with filamentous networks of hyalin that extend between microvilli and to the egg surface below (e.g., see Fig. 32).

Actin polymerization plays a central role in the growth of these microvilli. If one blocks polymerization by application of cytochalasin B before fertilization,

288

D.E. CHANDLER

by a number of investigators (Burgess and Schroeder,

1977; Chandler and Heuser, 1981; Spudich and Amos, 1979);frequently these cores extend into the egg proper appearing almost like rootlets (Fig. 35). The broad cytoplasmic sheets that connect several tips contain complex, interwoven networks of microfilaments (arrow, Fig. 36). Frequently, these microfilaments appear in groupings that are interconnected by shorter filaments (arrows, Fig. 37). It is now clear that both the calcium and pH signals work in concert to control microvillar growth. This has been shown experimentally by activating eggs with the calcium ionophore A23187 in sodium-free sea water (Begg et al., 1982; Carron and Longo, 1982). In this protocol the egg experiences a calcium signal but the pH signal is entirely eliminated because the Na+lH+ exchanger that produces this pH change requires external sodium ions. Eggs treated in this manner exhibit elongated microvilli that are filled with loose networks of actin filaments. These microvilli are flaccid, however, because their cores do not contain the bundles of parallel microfilaments seen in the thin, erect fingers of normal microvilli. Readdition of NaCl to the sea water, or of salts of weak bases such as NH,Cl, results in cytoplasmic alkalinization and formation of microfilament bundles within these microvilli. These observations suggest that the initial polymerization of actin that produces elongate microvilli requires only the calcium signal, while formation of microfilament bundles is regulated by the cytoplasmic pH increase. This conclusion is supported by data from Begg and Rebhun (1979) showing that formation of actin bundles in isolated egg cortices can be regulated by medium pH: substantial bundles are formed a t pH 7.5 while few are formed at pH 6.7. Overall, the growth of microvilli results in a three- to fourfold increase in F-actin that remains with the cortex during isolation (Bryan and Kane, 1978; Burgess and Schroeder, 1977; Spudich and Spudich, 1979). As suggested by Begg et al. (1982) and Tilney and Jaffe (19801, growth of microvilli after fertilization actually does take on the appearance of a two-step process. During the calcium signal microvillar growth is rapid but microvilli are relatively limp in appearance, while 1 to 2 min later during the pH signal microvilli become erect and contain microfilament bundles (Begg et al., 1982; Chandler and Heuser, 1981; Tilney and Jaffe, 1980; this study). Bundling of microfilaments has been found to be mediated by specific proteins (e.g., fimbrin in the intestinal brush border) and there is at Fig. 35. Most microvilli contain organized bundles of microfilaleast one candidate for this function in the sea urchin ments that appear to extend into the cytoplasm as anchoring egg cortex. Fascin, a 58 kD protein that causes forma“rootlets.” Specimen was quick-frozen 5 min postinsemination and tion of actin bundles in extracts of sea urchin egg freeze-substituted. x 120,000. (Bryan and Kane, 19781, has been shown to be present in the microvilli of fertilized sea urchin eggs (Otto et al., 1980). there is no growth and the egg is left with small, bulExactly what triggers the initial polymerization of bous micrivilli about the size of those on unfertilized actin just after fertilization is not known although it is eggs (Fig. 33; see also Longo, 1980). In thin sections of generally believed that the calcium signal starts the quick-frozen specimens the fertilized egg cortex con- chain of events. In this regard several calcium-regutains almost no organelles and is filled with cytoskel- lated proteins have been isolated that could influence etel elements (Fig. 34). The microvillar tips contain the polymerization state of actin. The first, sea urchin cores of bundled microfilaments that have been studied egg spectrin, a 2371234 doublet on SDS-PAGE gels, has

SEA URCHIN EGG CORTEX

Figs. 36 and 37. Microfilaments within the bases of microvilli exhibit criss-crossed networks (large arrow) and foci where filaments are connected by short linkers (small arrows). Specimen was quickfrozen at 5 min postinsemination and freeze substituted. (Reproduced from Chandler and Heuser, 1981, with permission of Academic Press.) x 180,000.

289

290

D.E. CHANDLER

been isolated by Fishkind et al. (1987) and rhodamineconjugated antibodies to fodrin, a member of the spectrin family, have been used to localize such a protein at the inner aspect of the egg plasma membrane (Schatten et al., 1986).Like other members of the family, egg spectrin has actin crosslinking ability and mixtures of the actin and egg spectrin are capable of forming gels; at certain spectrin concentrations, gel formation is inhibited at micromolar levels of calcium (Fishkind et al., 1987). Second, Chou and Rehbun (1986) have shown that sea urchin eggs contain a myosin light chain kinase activity that is calcium-calmodulin-dependent. Since the phosphorylation state of myosin in many nonmuscle cells regulates actin-myosin interaction (Adelstein and Conti, 1975; Adelstein et al., 1973) one could envision a situation in which calcium could regulate the contractile state of actin gels through myosin and myosin light chain kinase activity, much as is thought to be the case in platelets (Daniel et al., 1981).Consistent with this hypothesis is that phosphorylation of myosin light chains in sea urchin eggs accompanies contraction of actin gels in vitro (Burgess et al., 1984; Kane, 1983). Clearly, the exact molecular pathway by which the calcium signal restructures the egg cortex at fertilization is not known although we do know some of the potential players.

CONCLUSIONS The sea urchin egg represents an excellent example of how a set of intracellular signals coordinates a sequence of structural changes designed to prepare the cell for new functions. Fertilization is remarkably similar to other cell responses in which a dormant, metabolically inactive cell is reconfigured into a metabolically active, dividing cell. For example, fibroblasts and lympohcytes, upon stimulation with mitogens or growth factors, respond with an immediate but transient rise in cytosolic free calcium and a slower but longer lasting increase in cytosolic pH (Hesketh et al., 1985; Moolenaar et al., 1984; Rozengurt and Mendoza, 1985). Many organisms that undergo encystment resume activity and cell division with a marked cytosolic alkalinization (Busa and Nuccitelli, 1984). Thus, the intracellular signals elicited in the sea urchin egg at fertilization represent a communication paradigm that is common to vertebrate and mammalian cells. Sea urchin eggs, however, are unlike many other cells in that their responses to these signals are rapid and orderly, they exhibit both ultrastructural and biochemical consequences, and to a certain extent these responses can be studied independently of one another. Exocytosis is laid out spatially and temporally and is complete within 45 s. Endocytosis occurs with an extremely intense burst between 2 and 4 min. Microvillar growth occurs in sequential steps between 1and 5 min. Pronuclear formation, karyokinesis, and syngamy take place concurrently or soon thereafter, and these are followed by rapidly accelerating metabolic activities including respiration, protein synthesis, and DNA synthesis. It is not surprising then that fertilization in sea urchin eggs has interested cell biologists for well over

100 years, and that virtually every component of its response to activation has become a model system for detailed molecular study.

ACKNOWLEDGMENTS The original research described herein was supported by grants from NSF (DCB-8810200) and NIH (HD00619). REFERENCES Adelstein, R., and Conti, M.A. (1975) Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature, 256: 597-598. Adelstein, R.S., Conti, M.A., and Anderson, W. (1973) Phosphorylation of human platelet myosin. Proc. Natl. Acad. Sci. USA, 80: 3115-3119. Anderson, E. (1968) Oocyte differentiation in the sea urchin Arbacia punctulatu, with particular reference to the origin of cortical granules and their participation in the cortical reaction. J . Cell Biol., 37514-538. Anderson, E. (1970) A cytological study of the centrifuged whole, half, and quarter eggs of the sea urchin Arbacia punctulata. J. Cell Biol., 47:711-733. Begg, D.A., and Rebhun, L.I. (1979) pH regulates the polymerization of actin in the sea urchin egg cortex. J . Cell Biol., 83241-248. Begg, D.A., Rebhun, L.I., and Hyatt, H. (1982) Structural organization of actin in the sea urchin egg cortex: microvillar elongation in the absence of actin filament bundle formation. J. Cell Biol., 93: 24-32. Berridge, M.J. (1987) Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem., 56:159-194. Bonder, E.M., Fishkind, D.J., Cotran, N.M., and Begg, D.A. (1989) The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin. Dev. Biol., 134:327-341. Bryan, J . , and Kane, R.E. (1978) Separation and interaction of the major components of sea urchin actin gel. J. Mol. Biol., 125:207224. Burgess, D.R., and Schroeder, T.E. (1977) Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs. J. Cell Biol., 74:1032-1037. Burgess, D.R., Kane, R.E., and Yabkowitz, R. (1984) Myosin light chain phosphorylation regulates sea urchin actin gel contraction. J. Cell Biol., 99:34a. Busa, W., and Nuccitelli, R. (1984) Metabolic regulation via intracellular pH. Am. J . Physiol., 246:R409-R438. Carron, C.P., and Longo, F.J. (1982) Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg. Dev. Biol., 89:128-137. Chambers, E.L., and de Armendi, J . (1979) Membrane potential, action potential and activation potential of the eggs of the sea urchin Lytechinus uariegutus. Exp. Cell Res., 122203-218. Chambers, E.L., and Hinckley, R.E. (1979) Non-propagated cortical reactions induced by the divalent ionophore A23187 in eggs of the sea urchin, Lytechinus uariegatus. Exp. Cell Res., 124:441-446. Chandler, D.E. (1984a) Exocytosis involves highly localized membrane fusions. Biochem. SOC.Trans., 12961-963. Chandler, D.E. (1984b) Comparison of a fixed and quick frozen sea urchin eggs: exocytosis is preceded by a local increase in membrane mobility. J . Cell. Sci., 7223-36. Chandler, D.E. (1984~)Exocytosis in vitro: ultrastructure of the isolated sea urchin egg cortex as seen in platinum replicas. J . Ultrastruct. Res., 89:198-2 11. Chandler, D.E., and Heuser, J. (1979) Membrane fusion during secretion: cortical granule exocytosis in sea urchin eggs as studied by quick-freezing and freeze fracture. J. Cell Biol., 83:91-108. Chandler, D.E., and Heuser, J. (1980a) The vitelline layer of the sea urchin egg and its modification during fertilization: a freeze fracture study using quick freezing and deep etching. J. Cell Biol., 84:618-632. Chandler, D.E., and Heuser, J. (1980b) Arrest of membrane fusion events in mast cells by quick-freezing. J . Cell Biol., 86:666-674. Chandler, D.E., and Heuser, J . (1981) Postfertilization growth of microvilli in the sea urchin egg: new views from eggs that have been

SEA URCHIN EGG CORTEX quick-frozen, freeze-fractured, and deeply etched. Dev. Biol., 82: 393-400. Chandler, D.E., and Vacquier, V.D. (1988) Phorbol myristate acetate induces the phosphorylation of plasma membrane-associated proteins in sea urchin eggs. Growth Differ. Dev., 30:49-59. Chandler, D.E., Whitaker, M.J., and Zimmerberg, J . (1989) High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly. J. Cell Biol., 109:1269-1278. Chou, Y.-H., and Rebhun, L.I. (1986) Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependentkinase with myosin light chain phosphorylating activity. J . Biol. Chem., 261: 5389-5395. Ciapa, D.L., and Whitaker, M. (1986) Two phases of inositol polyphosphate and diacylglycerol production at fertilisation. FEBS Lett., 195:347-351. Clapper, D., and Lee, H.-C. (1985) Inositol triphosphate induces calcium release from nonmitochondrial stores in sea urchin egg homogenates. J . Biol. Chem., 260:13947-13954. Crossley, I., Swann, K., Chambers, E.L., and Whitaker, M. (1988) Activation of sea urchin eggs by inositol phosphates is independent of external calcium. Biochem. J., 252:257-262. Curran, M., Zimmerberg, J., and Cohen, F.S. (1988) Dynamics of exocytic pore formation in mast cells of the beige mouse. Biophys. J., 53:322a. Daniel, J.L., Molish, I.R., and Holmsen, H. (1981) Myosin phosphorylation in intact platelets. J . Biol. Chem., 256:7510-7514. David, C., Halliwell, J., and Whitaker, M.J. (1988) Some properties of the membrane currents underlying the fertilization potential in sea urchin eggs. J . Physiol. (Lond.), 402:139-154. Detering, N.K., Decker, G.L., Schmell, E.D., and Lennarz, W.L. (1977) Isolation and characterization of plasma membrane associated cortical granules from sea urchin eggs. J. Cell Biol., 75399-914. Dube, F., Schmidt, T., Johnson, C.H., and Epel, D. (1985) The hierarchy of requirements for a n elevated intracellular pH during early development of sea urchin embryos. Cell, 40:657-666. Eddy, E.M., and Shapiro, B.M. (1976) Changes in the topography of the sea urchin egg after fertilization. J . Cell Biol., 71:35-48. Eisen, A,, Kiehart, D.P., Wieland, S.J., and Reynolds, G.T. (1984) Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs. J . Cell Biol., 99:1647-1654. Epel, D. (1964) A primary metabolic change at fertilization: interconversion of pyridine nucleotides. Biochem. Biophys. Res. Commun., 17:62-68. Epel, D. (1978) Mechanisms of activation of sperm and egg during fertilization of sea urchin gametes. Curr. Top. Dev. Biol., 12:186246. Epel, D., Steinhardt, R., Humphreys, T., and Mazia, D. (1974) An analysis of the partial metabolic derepression of sea urchin eggs by ammonia; the existence of independent pathways. Dev. Biol., 40: 245-255. Epel, D., Patton, C., Wallace, R.W., and Cheung, W.Y. (1981) Calmodulin activates NAD kinase of sea urchin eggs: a n early event of fertilization. Cell, 23:543-549. Fisher, G.W., and Rebhun, L.I. (1983) Cortical granule exocytosis is followed by a burst phase of membrane retrieval via coated vesicles. Dev. Biol., 99:456-472. Fisher, G.W., Summers, R.G., and Rebhun, L.I. (1985)Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis. Dev. Biol., 109:489-503. Fishkind, D.J., Bonder, E.M., and Begg, D.A. (1987) Isolation and characterization of sea urchin egg spectrin: calcium modulation of the spectrin-actin interaction. Cell Motil. Cytoskel., 7:304-314. Foerder, C.A., Klebanoff, S.J., and Shapiro, B.M. (1978) Hydrogen peroxide production, chemiluminescence and the respiratory burst of fertilization: interrelated events in early sea urchin development. Proc. Natl. Acad. Sci. USA, 75:3183-3187. Gilkey, J.C. (1983)Roles of calcium and pH in activation of eggs of the Medaka fish, Oryzias latipes. J . Cell Biol., 97:669-678. Gilkey, J.C., Jaffe, L.F., Ridgeway, E.B., and Reynolds, G.T. (1978) A free calcium wave transverses the activating egg of the Medaka, Oryzias latipes. J. Cell Biol., 76:448-466. Gilman, A.G. (1987) G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem., 56:615-650. Glabe, C.G., and Vacquier, V.D. (1978) Egg surface glycoprotein receptor for sea urchin sperm bindin. Proc. Natl. Acad. Sci. USA, 75:881-885. Gomperts, B.D., Barrowman, M.M., and Cockcroft, S. (1986)Dual role

291

for guanine nucleotides in stimulus-secretion coupling: an investigation of mast cells and neutrophils. Fed. Proc., 45:2156-2161. Grainger, J.L., Winkler, M.M., Shen, S.S., and Steinhardt, R.A. (1979) Intracellular pH controls protein synthesis rate in the sea urchin egg and early embryo. Dev. Biol., 68:396-406. Grinstein, S., and Rothstein, A. (1986) Mechanisms of regulation of the Na/H exchanger. J . Membr. Biol., 9O:l-12. Haggerty, J.G., and Jackson, R.C. (1983) Release of granule contents from sea urchin egg cortices. J. Biol. Chem., 258:1819-1825. Hensen, J.H., and Begg, D. (1988) Filamentous actin organization in the unfertilized sea urchin egg cortex. Dev. Biol., 127:338-348. Hensen, J.H., Begg, D., Beaulieu, S.M., Fishkind, D.J., Bonder, E.M., Terasaki, M., Lebeche, D., and Kaminer, B. (1989) A calsequestrinlike protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo. J . Cell Biol., 109:149-162. Hesketh, T.R., Moore, J.P., Morris, J.D.H., Taylor, M.V., Rogers, J., Smith, G.A., and Metcalfe, J.C. (1985) A common sequence of calcium and pH signals in the mitogenic stimulation of eukaryotic cells. Nature, 313:481-484. Heuser, J., Reese, T.S., Dennis, M.J., J a n , Y., Jan, L., and Evens, L. (1979) Synaptic vesicle exocytosis captured by quick-freezing and correlated with quanta1 transmitter release. J . Cell Biol., 81:275300. Hinegardner, R.T., Rao, B., and Feldman, D.E. (1964) The DNA synthetic period during early development of the sea urchin egg. Exp. Cell Res., 36:53-61. Hiramoto, Y. (1974) Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage. Exp. Cell Res., 89: 320-326. Jackson, R.C., and Crabb, J.R. (1988) Cortical granule exocytosis in the sea urchin egg. Curr. Top. Membr. Trans., 32:45-87. Jackson, R.C., Ward, K.K., and Haggerty, J.G. (1985) Mild proteolytic digestion restores exocytotic activity to N-ethylmaleimide-inactivated cell surface complex from sea urchin eggs. J . Cell Biol., 101: 6-11. Jaffe, L.A. (1976) Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature. 261:68-71. Jaffe, L.A. (1986) Electrical regulation of sperm-egg fusion. Annu. Rev. Physiol., 48:191-200. Jaffe, L.A., and Robinson, K.R. (1978) Membrane potential of the unfertilized sea urchin egg. Dev. Biol., 62:215-228. Jaffe, L.A., Gould-Somero, M., and Holland, L.S. (1982) Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization. J . Cell Biol., 92:616-621. Jaffe, L.A., Turner, P.R., Kline, D., Kado, R.T., and Shilling, F. (1988) G-proteins and egg activation. In: Regulatory Mechanisms in Developmental Processes. G. Eguchi, T.S. Okada, and L. Saxen, eds. Elsevier Scientific Publishers, Ltd., Amsterdam, pp. 15-18. Jaffe, L.F. (1983) Sources of calcium in egg activation: a review and hypothesis. Dev. Biol., 99:265-276. Johnson, C.H., and Epel, D. (1981) Intracellular pH of sea urchin eggs measured by the dimethyloxalidinedione (DMO) method. J . Cell Biol., 89:284-291. Johnson, C.H., Epel, D., and Paul, M. (1987) Intracellular pH and activation of sea urchin eggs after fertilisation. Nature, 262:661664. Kamel, L.C., Bailey, J., Schoenbaum, L., and Kinsey, W. (1985) Phosphatidylinositol metabolism during fertilization in the sea urchin egg. Lipids, 20:350-356. Kane, R.E. (1983) Interconversion of structural and contractile actin gels by insertion of myosin during assembly. J . Cell Biol., 97:17451752. Kay, E.S., and Shapiro, B.M. (1985) The formation of the fertilization membrane of the sea urchin egg. In: Biology of Fertilization, Vol. 3 C.B. Metz and A. Monroy, eds. Academic Press, Orlando, pp. 45-81. Kidd, P. (1978) The jelly and vitelline coats of the sea urchin egg: new ultrastructural features. J . Ultrastruct. Res., 64:204-215. Kikkawa, U., and Nishizuka, Y. (1986) The role of protein kinase C in transmembrane signalling. Annu. Rev. Cell Biol., 2:149-178. Levitan, I.B. (1985) Phosphorylation of ion channels. J. Membr. Biol., 87:177-190. Longo F.J. (1980) Organization of microfilaments in sea urchin (Arbacia punctuluta) eggs at fertilization: effects of cytochalasin B. Dev. Biol., 74:422-433. Longo, F. (1989) Egg cortical architecture. In: The Cell Biology of Fertilization. H. Schatten and G. Schatten, eds. Academic Press, Orlando, pp. 108-138.

292

D.E. CHANDLER

Longo, F.J., Lynn, J.W., McCulloh, D.H., and Chambers, E.L. (1986) Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus uariegatus. Dev. Biol., 118:155-166. Lubbock, R., Gupta, B.L., and Hall, T.A. (1981) Novel role of calcium in exocytosis: mechanisms of nematocyst discharge as shown by x-ray microanalysis. Proc. Natl. Acad. Sci. USA, 78:3624-3628. Lynn, J.W., and Chambers, E.L. (1984) Voltage clamp studies of fertilization in sea urchin eggs. I. Effect of clamped membrane potential on sperm entry, activation, and development. Dev. Biol., 102: 98-109. Lynn, J.W., McCulloh, D.H., and Chambers, E.L. (1988) Voltage clamp studies of fertilization in sea urchin eggs. 11. Current patterns in relation to sperm entry, nonentry, and activation. Dev. Biol., 128:305-323. Mabuchi, I., and Sakai, H. (1972)Cortex protein of sea urchin eggs, its purification and other protein components of the cortex. Dev. Growth Differ., 14247-261. Majerus, P.W., Neufeld, E.J., and Wilson, D.B. (1984) Production of phosphoinositide-derived messengers. Cell, 37:701-703. Mazia, D. (1974) Chromosome cycles turned on in unfertilized sea urchin eggs exposed to NH,OH. Proc. Natl. Acad. Sci. USA, 71: 690-693. Merkle, C.J., and Chandler, D.E. (1989)Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening. J. Membr. Biol., 112:223-232. Mitchison, J.M., and Swann, M.M. (1955) The mechanical properties of the cell surface: the sea urchin egg from fertilization to cleavage. J. Exp. Biol., 32:734-750. Moolenaar, W.H., Tertoolen, L.G.J., and de Latt, S.W. (1984) Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature, 304:645-648. Moy, G.W., Kopf, G.S., Gache, C., and Vacquier, V.D. (1983) Calciummediated release of glucanase activity from cortical granules of sea urchin eggs. Dev. Biol., 100:267-274. Nishizuka, Y. (1984) The role of protein kinase C in the cell surface signal transduction and tumour promotion. Nature, 308:693-698. Nishizuka, Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature, 334:661-665. Oberdorf, J.A., Head, J.F., and Kaminer, B. (1986) Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis. J . Cell Biol., 102:2205-2210. Oberdorf. J.. Vilar-Roias. C., and Epel, D. (1989) The localization of PI and PIP kinase ac6vities in the-sea urchin egg and their modulation following fertilization. Dev. Biol., 131:236-242. Ornberg, R.L., and Reese, T.S. (1981) Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes. J . Cell Biol., 90: 40-54. Otto, J.J., Kane, R.E., and Bryan, J. (1980)Redistribution of actin and fascin in sea urchin eggs after fertilization. Cell Motil., 1:31-40. Poenie, M., and Epel, D. (1987) Ultrastructural localization of intracellular calcium stores by a new cytochemical method. J. Histochem. Cytochem., 35:939-956. Rossignol, D.P., Earles, B.G., Decker, G.L., and Lennarz, W.J. (1984) Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus. Dev. Biol., 104:308-321. Rozengert, E., and Mendoza, S.A. (1985) Synergistic signals in mitogenesis: role of ion fluxes, cyclic nucleotides, and protein kinase C in Swiss 3T3 cells. J. Cell Sci. Suppl., 3:229-242. Sardet, C. (1984) The ultrastructure of the sea urchin egg cortex isolated before and after fertilization. Dev. Biol., 105:196-210. Sardet, C., and Chang, P.(1987) The egg cortex: from maturation through fertilization. Cell Differ., 21:l-19. Sasaki, H., and Epel, D. (1983) Cortical vesicle exocytosis in isolated cortices of sea urchin eggs: description of a turbidometric assay and its utilization in studying effects of different media on discharge. Dev. Biol., 98:327-337. Schatten, G. (1981) Sperm incorporation, the pronuclear migrations, and their relation to the establishment of the first embryonic axis: time-lapse video microscopy of the movements during fertilization of the sea urchin Lytechinus uariegutus. Dev. Biol., 86:426-437. Schatten, G. (1982) Motility during fertilization. Int. Rev. Cytol., 79: 59-163. Schatten, H., and Schatten, G. (1980) Surface activity a t the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity. Dev. Biol., 78:435-449. Schatten, H., Cheney, R., Balczon, R., Willard, M., Cline, C., Simerly,

C., and Schatten, G. (1986) Localization of fodrin during fertilization and early development of sea urchins and mice. Dev. Biol., 118:l-10. Schroeder, T.E. (1978) Microvilli on sea urchin eggs: a second burst of elongation. Dev. Biol., 64:342-346. Schroeder, T.E. (1979) Surface area change at fertilization: resorption of the mosaic membrane. Dev. Biol., 70:306-326. Schroeder, T.E. (1985) Cortical expressions of polarity in the starfish oocyte. Dev. Growth Differ., 27:311-321. Schuel, H. (1985) Functions of egg cortical granules. In: Biology of Fertilization, Vol. 3. C.B. Metz and A. Monroy, eds. Academic Press, Orlando, pp. 1-44. Schmidt, T., Patton, C., and Epel, D. (1982) Is there a role for the Ca2+ influx during fertilization of the sea urchin egg? Dev. Biol., 90: 284-290. Shen, S.S. (1989a) N a + - H + antiport during fertilization of the sea urchin egg is blocked by W-7 but is insensitive to K252a and H-7. Biochem. Biophys. Res. Commun., in press. Shen, S.S. (1989b) Protein kinase C and regulation of the N a + / H + antiporter activity during fertilization of the sea urchin egg. In: Mechanisms of Egg Activation. R. Nuccitelli, G.N. Cherr, and W.H. Clark eds. Plenum Press, New York, pp. 173-199. Shen, S.S., and Burgart, L.J. (1985) Intracellular sodium activity in the sea urchin egg during fertilization. J. Cell Biol., 101:420-426. Shen, S., and Burgart, L.J. (1986) 1,2 Diacylglycerols mimic phorbol 12-myristate 13-acetate activation of the sea urchin egg. J. Cell Physiol., 127:330-340. Shen, S.S., and Steinhardt, R.A. (1978) Direct measurement of intracellular pH during metabolic derepression of the sea urchin egg. Nature, 272:253-254. Shen, S.S., and Steinhardt, R.A. (1979) Intracellular pH and the sodium requirement at fertilisation. Nature, 282:87-89. Shen, S., and Steinhardt, R.A. (1984) Time and voltage windows for reversing the electrical block to fertilization. Proc. Natl. Acad. Sci. USA, 81:1436-1439. Spudich, A., and Spudich, J.A. (1979) Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs. J . Cell Biol., 82:212-226. Spudich, J.A., and Amos, L.A. (1979) Structure of actin filament bundles from microvilli of sea urchin eggs. J. Mol. Biol., 129:319-331. Stapleton, C.L., Mills, L.L., and Chandler, D.E. (1985) Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores. J . Exp. Zool., 234:289-299. Steinhardt, R.A., and Epel, D. (1974) Activation of sea urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. USA, 71:1915-1919. Steinhardt, R.A., Shen, S.S., and Mazia, D. (1972) Membrane potential, membrane resistance and a n energy requirement for the development of potassium conductance in the fertilization reaction of echinoderm eggs. Exp. Cell Res., 72:195-203. Steinhardt, R., Zucker, R., and Schatten, G. (1977) Intracellular calcium at fertilization in the sea urchin egg. Dev. Biol., 58:185-196. Swann, K., and Whitaker, M. (1985) Stimulation of the Na/H exchanger of sea urchin eggs by phorbol ester. Nature, 314:274-277. Swann, K., and Whitaker, M. (1986) The part played by inositol triphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs. J . Cell Biol., 103:2333-2342. Swann, K., Ciapa, B., and Whitaker, M. (1987) Cellular messengers and sea urchin activation. In: Molecular Biology of Invertebrate Development. J.D. OConner, ed. Alan R. Liss, Inc., New York, pp. 45-69. Tilney, L.G., and Jaffe, L.A. (1980) Actin, microvilli and the fertilization cone of sea urchin eggs. J. Cell Biol., 87:771-782. Trimmer, J.S., and Vacquier, V.D. (1986) Activation of sea urchin gametes. Annu. Rev. Cell Biol., 2:l-26. Tsien, R.Y., and Poenie, M. (1986) Fluorescence ratio imaging: a new window into intracellular ionic signalling. Trends Biochem. Sci. 11:450-455. Turner, P.R., and Jaffe, L.A. (1989) G-proteins and the regulation of oocyte maturation and fertilization. In: The Cell Biology of Fertilization. H. Schatten and G. Schatten, eds., Academic Press, Orlando, pp. 298-318. Turner, P.R., Sheetz, M.P., and Jaffe, L.A. (1984) Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature, 310:414-415. Turner, P.R., Jaffe, L.A., and Fein, A. (1986) Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-triphosphate and GTP-binding protein. J. Cell Biol., 102:70-76. Uhing, E.J., Propic, V., Jiang, H., and Exton, J.H. (1986) Hormone-

SEA URCHIN EGG CORTEX stimulated polyphosphoinositide breakdown in rat liver plasma membranes. J . Biol. Chem., 261:2140-2146. 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. (1976) Isolated cortical granules: a model system for studying membrane fusion and calcium-mediated exocytosis. J . Supramol. Struct., 527-35. Vacquier, V.D. (1981) Dynamic changes of the egg cortex. Dev. Biol., 84:l-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. Wang, Y.L., and Taylor, D.L. (1979) Distribution of fluorescently labelled actin in living sea urchin eggs during early development. J . Cell Biol., 82:672-679. Whitaker, M.J., and Aitchison, M.J. (1985) Calcium-dependent polyphosphoinositide hydrolysis is associated with exocytosis in vitro. FEBS Lett., 182:119-124. Whitaker, M., and Baker, P.F. (1983) Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function. Proc. R. SOC.Lond., B218:397-413. Whitaker, M.J., and Irvine, R.F. (1984) Inositol 1,4,5-trisphosphate microinjection activates sea urchin eggs. Nature, 312:636-639. Whitaker, M.J., and Steinhardt, R.A. (1981)The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs. Cell, 2595-103.

293

Whitaker, M.J., and Steinhardt, R.A.(1983) Evidence in support of the hypothesis of an electrically mediated fast block to polyspermy in sea urchin eggs. Dev. Biol., 95244-248. Whitaker, M.J., and Steinhardt, R.A. (1985) Ionic signalling in the sea urchin egg a t fertilization. In: Biology of Fertilization, Vol. 3. C.B. Metz and A. Monroy, eds. Academic Press, Orlando, pp. 167221. Whitaker, M.J., Swann, K., and Crossley, I. (1989) What happens during the latent period at fertilization. In: Mechanisms of Egg Activation. R. Nuccitelli, G.N. Cherr, and W.H. Clark, Jr., eds. Plenum Press, New York, pp. 157-171. Whitaker, M.J., and Zimmerberg, J . (1987) Inhibition of secretory granule discharge during exocytosis in sea urchin eggs by polymer solutions. J . Physiol. (Lond.), 389527-539. Winkler, M.M., Steinhardt, R.A., Grainger, J.L., and Minning, L. (1980) Dual ionic controls for the activation of protein synthesis at fertilization. Nature, 287:558-560. Zimmerberg, J., Sardet, C., and Epel, D. (1985) Exocytosis of sea urchin egg cortical vesicles in vitro is retarded by hyperosmotic sucrose: kinetics of fusion monitored by quantitative light-scattering microscopy. J . Cell Biol., 101:2398-2410. Zimmerberg, J., Curran, M., Cohen, F.S., and Brodwick, M. (1987) Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc. Natl. Acad. Sci. USA, 84:15851589.