sterol complexes in fertilized and unfertilized sea urchin egg membranes

DEVELOPMENTAL

BIOLOGY

99,482-488

(1983)

Filipin/Sterol

Complexes in Fertilized and Unfertilized Sea Urchin Egg Membranes CHRISTOPHER

Department

P. CARRON~ AND FRANK J. LONGO

of Anatomy, University of Iowa, Iowa City, Iowa 52242

Received March 24, 1985; accepted in revised form

May

9, 1988

Sea urchin (Arbacia punctu.!uta) eggs and zygotes were treated with filipin in an effort to examine changes in membrane sterols at fertilization. The plasma membrane of treated unfertilized eggs possessed numerous fdipin/sterol complexes, while fewer complexes were associated with membranes delimiting cortical granules, demonstrating that the plasmalemma is relatively rich in fl-hydroxysterols in comparison to cortical granule membrane. Following fusion with the plasmalemma, membrane formerly delimiting cortical granules underwent a dramatic alteration in sterol composition, as indicated by a rapid increase in the number of filipin/sterol complexes. In contrast, portions of the zygote plasma membrane, derived from the plasmalemma of the unfertilized egg, displayed little or no change in filipin/sterol composition. Other than regions of the plasma membrane engaged in endocytosis, the plasmalemma of the zygote possessed a homogeneous distribution of filipin/sterol complexes and appeared similar to that of the unfertilized egg. These results demonstrate that following its fusion with the egg plasmalemma, membranes, formerly delimiting cortical granules, undergo a dramatic alteration in sterol composition. Changes in the localization of filipin/ sterol complexes are discussed in reference to alterations in egg plasmalemmal function at fertilization.

1979a,b; Longo, 1981, 1982; Wolf et al, 1981). Although lipids from surface ghosts, prepared from sea urchin eggs before and after fertilization have been analyzed (Barber and Mead, 1975), as far as we are aware, identification of sterol domains, as reported for sperm, have not been published for unfertilized or fertilized ova (cf. Friend, 1982). As a part of a series of investigations examining membrane changes at fertilization and their possible relation to functional changes at fertilization/activation, we have initiated experiments examining the distribution of sterols in unfertilized and fertilized sea urchin eggs. The membrane-perturbing effect of filipin has been utilized to visualize filipin/sterol complexes at the ultrastructural level of observation in a variety of cells (De Kruijff and Demel, 1974; Elias et a& 1979; Friend and Bearer, 1981). Previous studies have shown that filipin interacts stoichiometrically with p-hydroxysterols resulting in the formation of 20 to 25nm membrane protuberances visible in freeze-fracture replicas and in thin-sectioned material prepared for electron microscopy. These reproducible patterns of membrane protuberances provide a novel cytochemical tool for ultrastructural investigation of plasma membrane changes at fertilization.

INTRODUCTION

At fertilization, membranes formerly delimiting cortical granules, the sperm, and the egg integrate to form a continuous lamina that constitutes the plasma membrane of the zygote. Thus, with respect to its origin, the plasma membrane of the fertilized egg is a mosaic structure (Monroy and Moscona, 1979). Recent investigations have shown that the sperm and egg plasma membranes and the cortical granule membrane are morphologically distinct (Longo, 1981, 1982). These observations beg the question of whether or not this diversity persists following gamete fusion and the cortical granule reaction. That is, do identifiable membrane domains persist within the plasma membrane of the fertilized egg that were derived from the cortical granule and the spermatozoon? Moreover, where membrane domains do exist, is their structure and composition related to specific changes that occur as a result of egg activation? Alterations in the composition of the egg plasma membrane at fertilization have been reported with regard to lipids, proteins, and carbohydrates, indicating both a restriction and a diffusion of components within the plane of the zygote plasma membrane (Yanagimachi et aL, 1973; Eddy and Shapiro, 19’76; Johnson and Edidin, 1978; Campisi and Scandella, 1978, 1980; Gabel et aL, i Present address: Department Medicine, Houston, Tex. 77030.

A&a&a

of Cell Biology, Baylor College of

0012-1606/83 $3.00 Copyright All rights

MATERIALS

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

wnctulata,

logical Laboratory, 482

AND METHODS

obtained from the Marine BioWoods Hole, Massachusetts or Gulf

CARRON AND LONGO

Redistribution

Specimens Company, Panacea, Florida, were maintained in an ocean aquarium containing artifical seawater (ASW; Instant Ocean Aquarium Systems, Eastlake, Ohio). Animals were induced to spawn with a O&ml injection of 0.5 M KC1 solution (Costello et al, 1957). The eggs were shed into ASW and the sperm were collected “dry.” After washing with ASW, the eggs were fertilized and allowed to develop at 20°C with constant stirring. Eggs were collected at 20, 40, and 60 set and then at 1-min intervals up to 10 min postinsemination, fixed for 1 hr at 0°C in ASW containing 3% glutaraldehyde, 7% sucrose, and 1% acrolein. Specimens were washed for 1 hr at 0-4°C in ASW and then incubated in 300 pM filipin in ASW. Filipin (Upjohn Co., Kalamazoo, Mich.) was initially solubilized in dimethylsulfoxide (DMSO) and then added to ASW. To assesseffects of DMSO, control experiments with 0.5% DMSO but without filipin were performed. Specimens were incubated in filipin for 36 hr at 4°C and washed in ASW. Aliquots from each sample were processed for freezefracture replication or were postfixed in 1% OsOl, 0.75% KFez (CN), dissolved in ASW. Osmicated specimens were embedded in Spurr embedding medium, sectioned, and examined in a Philips 300 electron microscope. Specimens for freeze-fracture replication were prepared by the slow addition of glycerol to a final concentration of 20%. The samples were pipetted into a complementary replica device for fracturing, and frozen in liquid Freon 22 cooled with liquid nitrogen. Specimens were fractured in a Balzers BAF 301 apparatus at -130°C and replicated using an electron beam evaporation unit for platinum shadowing mounted at 45”. Carbon-platinum replicas were cleaned with household bleach, mounted on 300-mesh grids, and examined in a Philips 300 electron microscope. RESULTS

Freeze-fracture replicas of the cortex of Arbacia eggs treated with filipin demonstrated a distinctive pattern of protuberances, characteristic of filipin/sterol complexes observed in other cells (Figs. 1, 2; cf. Friend, 1982). Filipin/sterol complexes induced numerous indentations which were distributed uniformly throughout the plasma membrane; little or no difference was apparent between villous and microvillous regions (Figs. 1,2). Few or no filipin/sterol complexes were associated with membranes of the annulate lamellae, Golgi complexes, mitochondria, and female pronucleus. The membrane delimiting yolk granules possessed numerous protuberances (Fig. 1). In contrast to yolk bodies and the plasma membrane, relatively few filipin/sterol complexes were associated with membranes of cortical granules (Fig. 1). Filipin/sterol complexes of cortical

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granule membranes were, for the most part, localized along that region of the granule subjacent to the plasma membrane. In addition, elongate indentations were observed along the E and P faces of the cortical granule membrane, possibly reflecting a linear association of 6lipin/sterol complexes (Fig. 1). Because of the disruption induced by filipin in plasma and cortical granule membranes, reliable quantitation of complexes proved difficult to perform. In thin sections of unfertilized eggs, filipin/sterol complexes appeared as a general scalloping along the microvillous and nonvillous portions of the plasma membrane (Figs. 3,4). Only that portion of the cortical granule membrane subjacent to the plasma membrane exhibited a scalloped appearance; the remainder of the cortical granule membrane was unruffled. Inspite of differences in the number of protuberances, the inner and outer leaflets of the plasma and cortical granule membranes were equidistance throughout their lengths; dilations within the midregion of the plasma and cortical granule membranes treated with filipin were not observed (Figs. 3,4). The plasma membranes of specimens treated only with DMSO did not possessprotuberances or corrugations. Eggs fixed soon after the mixing of the gametes demonstrated stages of the cortical granule reaction and transformations in the morphology of cortical granule membranes (Figs. 5, 6). Because of their cup-like appearance and association with dispersing cortical granule material, portions of the plasma membrane of fertilized eggs formerly delimiting cortical granules were apparent up to 2 min postinsemination (Longo, 1981). Unlike the situation observed in unfertilized eggs, the membrane of dehiscing cortical granules possessednumerous filipinisterol complexes (Figs. 5-7). In freeze fracture replicas of dispersing cortical granules, domains, free of filipin/sterol complexes, were not apparent along the plasmalemma of activated eggs (Figs. 6, 7). In thin sections, the cortical granule membrane had a highly corrugated profile similar to that of the unfertilized, egg plasmalemma (Fig. 7). A wave of transition within the membrane of dehiscing cortical granules, i.e., the progressive accumulation of filipin/sterol complexes with membrane derived from cortical granules was not apparent. Filipin/sterol complexes were uniformly distributed throughout the zygote plasma membrane; elongate microvilli, as well as intervillous regions of the plasmalemma demonstrated a corrugated appearance similar to that observed in the plasma membrane of the unfertilized egg (Fig. 8). Only those regions of the zygote plasma membrane undergoing endocytosis demonstrated an absence of filipin/sterol complexes (Figs. 8-11). Coated pits and vesicles lacked the corrugations characteristic of other portions of the zygote plasma

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membrane throughout their invagination poration into the cortical cytoplasm.

Redistribution

and incor-

DISCUSSION

The observations presented herein demonstrate differences in the plasma and cortical granule membranes of Arbacia eggs fixed with glutaraldehyde and acrolein and treated with filipin. Relative to the plasmalemma, the cortical granule membrane was associated with fewer filipin/ sterol complexes; most were present along the apical pole of the cortical granule, in association with the plasma membrane. Unlike the cortical granule membrane, the egg plasmalemma did not demonstrate specific domains and possessed a uniform distribution of complexes. Consequently, microvillous and nonmicrovillous membranous regions of both fertilized and unfertilized eggs were structurally similar to one another. Numerous laboratories have demonstrated that filipin induces the formation of pits and protrusions within membranes due to its interaction with sterols (Tillack and Kinsky, 1973; Kitajima et aL, 1976; De Kruijff and Demel, 1974; Brittman, 1978; Norman et aL, 1976; Robinson and Karnovsky, 1980; Friend and Bearer, 1981; Elias et aL, 1979). The aggregation of sterols into filipin/ sterol complexes is thought to be oriented either in the hydrophobic portion of the membrane (DeKruijff and Demel, 1974; Robinson and Karnovsky, 1980), or to the side of the membrane (Friend and Bearer, 1981; Elias et ak, 1979). In the present study protuberances and invaginations were invariably bilayers in thin section images; in no instance did we detect a widening of the hydrophobic core of the membrane. The distinction between the plasmalemma and cortical granule membrane was consistent from specimen to specimen and filipin/sterol complexes were associated with organelles (e.g., yolk bodies) in a uniform manner despite their position within the egg cytoplasm. Therefore, differences observed between the plasma and the

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cortical granule membranes are believed to represent an inequality in sterol content. However, we have not eliminated the possibility that these differences may be due to other factors, such as a masking of sterols within membranes, an insensitivity of the technique, focal membrane impermeability to filipin, or to the lack of deformability owing to structures associated with the membrane (Norman et aL, 1976; Elias et ah, 1979). Observed differences in the presence of filipin/sterol complexes in the various membranous systems of the egg indicate the capability of the cell to sequester its membrane components into different groupings. Differences have also been described in other cells, particularly the guinea pig spermatozoon where specific domains are recognizable in the plasmalemma (Friend, 1980, 1982; Friend and Bearer, 1981). The localization of sterols to the apical pole of the cortical granule membrane indicates that there are constraints to the lateral diffusion of specific lipids. This localization may be affected by a member of parameters, including intramembranous components and cytoskeletal elements in association with the membrane (Friend, 1980; Orci et al., 1980). These results differ from those recently published, indicating that the cholesterol content of the cortical granule is significantly higher than that of the plasma membrane in Lytechinus variegatus eggs (Decker and Kinsey, 1983). Based on these data, Decker and Kinsey (1983) suggested that plasma membrane cholesterol levels would be substantially increased following the cortical granule reaction, a finding they were unable to confirm. We are unable to reconcile discrepancies between this and the present investigation, although they may be related to differences in species and methodologies. AS indicated herein the plasma membrane of the zygote did not demonstrate domains lacking filipin/sterol complexes corresponding to the cortical granule membrane. The absence of such features suggests that the cortical granule membrane is significantly altered when

FIG. 1. Freeze-fracture replica of the cortex of a filipin-treated, unfertilized Arbucia egg. The plasmalemma and membrane delimiting volk bodies (YB) possess numerous filipin/sterol complexes. The membranous protrusions along microvilli (MV) are due to the presence of flipin/ sterol complexes. Relatively fewer filipin/sterol complexes are associated with the membrane surrounding cortical granules (CG). Many of the complexes associated with the membrane of cortical granules are located along the region subjacent to the plasma membrane (arrows). Elongate indentations (I) are also present within the membrane delimiting cortical granules. X46,500. FIG. 2. Arbucia egg demonstrating numerous protrusions (arrows) due to the formation of filipin/sterol complexes. MV, microvilli. X32,000. FIGS. 3 AND 4. Thin sections of filipin-treated unfertilized eggs corresponding to images seen in freeze-fracture replicas. The plasma membrane is plicated along microvillous (MV) and nonmicrovillous portions of the egg surface, indicating that it is relatively rich in sterols. Notice that membranes delimiting the cortical granules (CG) demonstrate a rather smooth, uniform profile and are plicated only in areas immediately subjacent to the plasmalemma (small arrows). The plications of the plasma membrane at the large arrows indicate that the complexes are present immediately adjacent to the plasmalemma and not within the hydrophobic region of the membrane. The corrugated appearance demonstrated by portions of the cortical granule membranes indicate a similar relation. Fig. 3, X40,000, Fig. 4, X75,000. FIG. 5. Dehiscing cortical granule of a fertilized egg treated with filipin. The membrane delimiting the granule is highly plicated (arrows) as is the plasma membrane (PM). DM, dispersing cortical granule material. X64,100.

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cosity and molecular motion of lipids in membranes; they have been considered as membrane stablizers (Papahadjopoulos et al, 1973; Singer, 1976; Chen et al, 1978). The filipin/sterol complexes along that portion of the cortical granule membrane subjacent to the plasmalemma, are situated in an area corresponding to specializations of the plasma membrane previously described (Longo, 1981). Whether or not, and how these two areas are related to one another, has not been determined. The presence of sterols along the apical pole of cortical granules may function in the regulation of the cortical granule release. Friend and Bearer (1981) indicated that where fusion between the outer acrosomal and plasma membrane occur, foci within the membrane clear of both filipin/sterol complexes and intramembranous particles. They suggested that sterols and proteins move from the hydrophobic core of apposed membranes, prior to their fusion (cf. also Davis, 1980). Similar processes may also occur at the time of the cortical granule reaction. The presence of coated pits and vesicles lacking plications seen in other portions of the zygote plasma membrane indicate the establishment of specific domains that were not present in the unfertilized egg. Whether this process involves the incorporation of components derived from the former plasma or cortical granule membranes or both was not established. Nevertheless, these observations indicate that, unlike the unfertilized egg, the plasma membrane of the zygote developed specific domains, low in sterols to allow detection by the formation of filipin complexes. Similar findings have also been reported for endocytosis in somatic cells (Montesano et al, 1979). Coated pits and vesicles represent membrane areas depleted in sterols, possibly in order to attain the fluidity required for endocytosis (Davis, 1980; Legault et al, 1979; Chapman, 1973; Jain, 1975; Demel and De Kruijiff, 1976; Montesano et al., 1979). Localized increases in membrane fluidity may be important not only for deformability of the plasma membrane associated with endocytosis but also for membrane processes leading to the formation of coated vesicles. Hence, the membranous region occupied by coated pits may represent areas having been “filtered”

it fuses with the plasmalemma. How this alteration is achieved has not been determined, although a number of possibilities are likely, including the lateral displacement of sterols from the “original” egg plasma membrane and an unmasking of sterols within the former cortical granule membrane. Similar schemes have been proposed for the increase in filipin/sterol complexes observed in the postacrosomal membrane following the acrosomal reaction (Friend, 1982). No evidence was found substantiating a lateral movement of sterols from the “original” egg plasma membrane immediately following the initiation of the cortical granule reaction, suggesting that if such a phenomenon did exist, it would involve a rapid diffusion of sterols. Previous investigations by Trauble and Sackmann (1972) have demonstrated the rapid diffusion of sterols in bilayers, which would account for our inability to “stop” the lateral movement of the sterol groups into membrane patches derived from cortical granules. The observations presented here are consistent with previous investigations examining the integration of membrane components in fertilized eggs by an analysis of intramembranous particle distribution and concanavalin A receptor localization (Longo, 1981,1982). These studies demonstrated a mixing of membrane components, derived from the original egg plasma membrane, into the former sperm plasmalemma and cortical granule membrane. Hence, in these cases definite domains were not apparent within the plasmalemma of the zygote that could be ascribed to their specific origin (cf. also Chandler and Heuser, 1979; Yanagimachi et al., 1973; Johnson and Edidin, 1978; Wolf et al, 1981). In conjunction with these studies, the present investigation indicates the plasticity of the plasma membrane of the fertilized egg with respect to sterols. Alterations in the cortical granule membrane intercalated into the plasma membrane during the cortical granule reaction may affect membrane function, and, hence, might be responsible for changes characteristic of the fertilized egg (Monroy and Moscona, 1979). Cholesterol inhibits the permeability of membranes and the ability of phospholipids to activate preparations of (Nat + K+) ATPase. Sterols may also affect the internal vis-

FIG. 6. Freeze-fracture replica of a dehiscing cortical granule, 40 set postinsemination. The membrane, formerly lining the cortical granule (arrows), is associated with numerous filipin/sterol complexes. An asterisk is located in the center of the dehiscing granule. MV, microvilli. x77,000. FIG. 7. Intact (CG) and dehiscing (*) cortical granules of a fertilized egg, 40 set postinsemination. The arrows indicate the membrane of the dehiscing cortical granule which is highly plicated. In contrast, the membrane of the intact cortical granule has a relatively smooth contour. DM, dehisced cortical granule material. ~44,000. FIG. 8. Surface of a filipin-treated, fertilized egg in which microvillous and nonmicrovillous regions of the plasma membrane demonstrate numerous plications due to the presence of filipin/sterol complexes (arrows). Domains within the plasmalemma of fertilized eggs, free of fllipin/sterol complexes, and reminiscent of the cortical granule membrane, are not observed. The coated pit at the base of the microvillus lacks filipin/sterol complexes. ~55,000. FIGS. 9-11. Stages of endocytosis in filipin-treated, fertilized eggs. Filipin/sterol complexes are associated with the plasma membrane except at those regions of forming endocytotic vesicles. Fig. 9, X70,000; Fig. 10, X42,000; Fig. 11, X67,500.

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of most sterols (Bretscher, 1976; Pearse, 1976; Altstiel and Branton, 1983). As a consequence, the internalized membrane would possess a sterol/phospholipid ratio relatively lower than that of the plasma membrane, a relationship that has been verified by the absence of filipin/sterol complexes described herein and supported by previous studies (Pearse, 1976; Montesano et aL, 1979; Altstiel and Branton, 1983). The technical assistance of Fred So is greatly appreciated. work was supported by funds from the NSF and the NIH.

This

REFERENCES ALTSTIEL, L., and BRANTON, D. (1983). Fusion of coated vesicles with lysosomes: Measurement with a fluorescence assay. CeU 32.921-929. BARBER, M. L., and MEAD, J. F. (1975). Comparison of lipids of sea urchin egg ghosts prepared before and after fertilization. Wilhelm Rwux’s Arch. 177,19-27. BITTMAN, R. (1978). Sterol-polyene antibiotic complexation: Role of membrane structure. Lipids 13, 686-691. BRETSCHER,M. S. (1976). Directed lipid flow in cell membranes. Nature (-) 266, 21-23. CAMPISI, J., and SCANDELLA, C. J. (1978). Fertilization-induced changes in membrane fluidity of sea urchin eggs. Science 199, 1336-1337. CAMPISI, J., and SCANDELLA, C. J. (1980). Bulk membrane fluidity increases after fertilization or partial activation of sea urchin eggs. J. BioL Ch.em 255, 5411-5419. 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. CeU BioL 83, 35-48. CHAPMAN, D. (1973). In “Biological Membranes” (D. Chapman and D. F. H. Wallach, eds.), Vol. 2, pp. 91-144. Academic Press, New York. CHEN, H. W., KANDUTSCH, A. A., and HEINIGER, H. J. (1978). Membrane anomalies in tumor cells. Prop. Exp. Tumor Res. 22,299-316. COSTELLO,D. P., DAVIDSON, M. E. EGGERS, A., Fox, M. H., and HENLEY, C. (1957). “Methods for Obtaining and Handling Marine Eggs and Embryos.” Lancaster Press, Lancaster, Pa. DAVIS, B. K. (1980). Interaction of lipids with the plasma membrane of sperm cells. I. The antifertilization action of cholesterol. Arch AndroL 5.249-254. DECKER, S. J., and KINSEY, W. H. (1983). Characterization of cortical secretory vesicles from the sea urchin egg. Dev. BioL 96, 37-45. DE KRUIJFF, B., and DEMEL, R. A. (1974). Polyene antibiotic-sterol interactions in membranes of Acholepbma laidlawii and lecithin lysosomes. III. Molecular structure of the polyene antibiotic-cholesterol complexes. Biochim Biophys. Acta 339,57-70. DEMEL, R. A., and DE KRUIJFF, B. (1976). The function of sterols in membranes. Biochim Biophys. Acta 457,109-132. EDDY, E. M., and SHAPIRO, B. M. (1976). Changes in the topography of the sea urchin egg after fertilization. J. Cell Bid 71, 35-48. ELIAS, P. M., FRIEND, D. S., and GOERKE, J. (1979). Membrane sterol heterogeneity. Freeze-fracture detection with saponins and filipin. J. Histochem C&o&em 27.1247-1260. FRIEND, D. S. (1980). Freeze-fracture alterations in guinea pig sperm membranes proceeding gamete fusion. In “Membrane-Membrane Interactions” (N. B. Gilula, ed.), pp. 153-165. Raven Press, New York. FRIEND, D. S. (1982). Plasma-membrane diversity in a highly polarized cell. J. Cell BioL 93, 243-249. FRIEND, D. S., and BEARER, E. L. (1981). (3-Hydroxysterol distribution as determined by freeze-fracture cytochemistry. Histochem J. 13, 535-546. GABEL, C. A., EDDY, E. M., and SHAPIRO, B. M. (1979a). After fertil-

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ization, sperm surface components remain as a patch in sea urchin and mouse embryos. CeU l&207-215. GAB% C. A., EDDY, E. M., and SHAPIRO, B. M. (1979b). Persistence of sperm surface components in the early embryo. In “The Spermatozoon” (D. W. Fawcett and J. M. Bedford, eds.), pp. 219-229. Urban & Schwarzenberg, Baltimore. JAIN, M. K. (1975). Role of cholesterol in biomembranes and related systems. Curr. Top. Membr. Transp. 6, l-57. JOHNSON,M., and EDIDIN, M. (1978). Lateral diffusion in plasma membrane of mouse egg is restricted after fertilization. Nature (Londan) 272,448-450. KITAJIMA, Y., SEKIYA, T., and NOZAWA, Y. (1976). Freeze-fracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasma membranes of Epidermophyton, Saccharomyces and red blood cells. A proposal of models for polyeneergosterol complex-induced membrane lesions. Biochim Biqphys. Acta 445,452-465. LEGAULT, Y., BLEUA, G., CHAPDELAINE, A., and ROBERTS, K. D. (1979). The binding of sterol sulfates to hamster spermatozoa. Steroids 34, 89-99. LONGO, F. J. (1981). Morphological features of the surface of the sea urchin (Arbacia pun&data) egg: Oolemma-cortical granule association. Den. Biol 84, 173-182. LONGO, F. J. (1982). Integration of sperm and egg plasma membrane components at fertilization. Den. BioL 89, 409-416. MONROY, A., and MOSCONA, A. A. (1979). “Introductory Concepts in Developmental Biology.” Univ. Chicago Press, Chicago. MONTESANO, R., PERRELET, A., VASSALLI, P., and ORCI, L. (1979). Absence of filipin-sterol complexes from large coated pits on the surface of cultured cells. Proc. NatL Ad Sci USA 76,639&-6395. NORMAN, A. W., SPIELVOGEL, A. M., and WONG, R. G. (1976). Polyene antibiotic-sterol interaction. A&J. Lipid Rex 14, 127-170. ORCI, L., MILLER, R. G., MONTESANO, R., PERRELET, A., AMHERDT, M., and VASSALI, P. (1980). Opposite polarity of Illipin-induced deformations in the membrane of condensing vacuoles and zymogen granules. Science 210, 1019-1021. PAPAHADJOPOULOS,D., COWDEN, M., and KIMELBERG, H. (1973). Role of cholesterol in membranes effects on phospholipid-protein interactions, membrane permeability and enzymatic activity. Biochim Biophys Ada 330,8-26. PEARSE, B. M. F. (1976). Clathrin: A unique protein associated with intracellular transfer of membrane by coated vesicles. Proc. NatL Acad. Sci USA 73.1257-1259. ROBINSON,J. M., and KARNOVSKY,M. J. (1980). Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J. Histochem C&&em 28, 161-168. SINGER, S. J. (1976). The fluid mosaic model of membrane structure: Some applications to ligand-receptor and cell-cell interactions. In “Surface Membrane Receptors” (R. A. Bradshaw, W. A. Frazier, R. C. Merel, D. I. Gottlieb, and R. A. Horigne-Angeletti, eds.), pp. l-24. Plenum Press, New York. TILLACK, T. W., and KINSKY, S. C. (1973). A freeze-etch study of the effects of filipin on liposomes and human erythrocyte membranes. B&him. Biophys. Actu 323,43-54. TROUBLE, H., and SACKMANN, E. (1972). Studies of the crystallineliquid crystalline phase transition of lipid model membranes. III. Structure of a steroid-lecithin system below and above the lipidphase transition. J. Amer. Chem. Sot 94, 4499-4510. WOLF, D. E., KINSEY, W., LENNARZ, W., and EDIDIN, M. (1981). Changes in the organization of the sea urchin egg plasma membrane upon fertilization: Indications from the lateral diffusion rates of lipidsoluble fluorescent dyes. Deu. Bid 81, 133-138. YANAGIMACHI, R., NICOLSON, G. L., NODA, Y. D., and FUJIMOTO, M. (1973). Electron microscopic observations of the distribution of acid anionic residues on hamster spermatozoa and eggs before and during fertilization. J. ultrastrud. Rea 43, 344-353.