Fertilization in Fucus

Summary Methods are described for the collection, treatment and uniform discharge of large quantities of gametes, and a measure of fertilization control in the monoecious brown alga, Fucus distichus. Fertilization was examined using the techniques of blister-formation, gamete separation experiments, and electron microscopy. Sperm enter the freshly-discharged egg packets through a mesochite pore and juxtaposition with the eggs early. However, experimental and thinsection data indicate that fertilization does not occur until the eggs dissociate from the mesochite and round up. Hence, the egg surface appears to undergo three functional changes following its release from the thallus: (1) a pre-dissociation state which inhibits fertilization within the mesochite; (2) a dissociation state when fusion of gametes is possible; and (3) a post-fertilization state characterized by the formation of extraneous coats. As the egg is activated by the sperm a nonmembranous layer appears to detach from the egg surface to form an activation layer. This is augmented by fibrous units to function as a fertilization barrier which ultimately thickens to form the cell wall. The area between the eggs within the egg packet is characterized by the presence of fibrous and particulate substances which are continuously given off through the egg surface. These apparently play a functional role in the sequential stages of fertilization in Fucus. These observations are discussed in terms of analogies with fertilization in the sea urchin.This work was supported by a Lalor Foundation Research Fellowship at Friday Harbor Biological Laboratories, University of Washington, and by a National Science Foundation Science Faculty Fellowship to the Swiss Federal Institute of Technology, Zürich, Switzerland.     

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.     

The role of jelly coats in sperm-egg encounters, fertilization success, and selection on egg size in broadcast spawners

Sperm limitation may be an important selective force influencing gamete traits such as egg size. The relatively inexpensive extracellular structures surrounding many marine invertebrate eggs might serve to enhance collision rates without the added cost of increasing the egg cell. However, despite decades of research, the effects of extracellular structures on fertilization have not been conclusively documented. Here, using the sea urchin Lytechinus variegatus, we remove jelly coats from eggs, and we quantify sperm collisions to eggs with jelly coats, eggs without jelly coats, and inert plastic beads. We also quantify fertilization success in both egg treatment groups. We find that sperm-egg collision rates increase as a function of sperm concentration and target size and that sperm are not chemotactically attracted to eggs nor to jelly coats in this species. In fertilization assays, the presence of the jelly coat is correlated with a significant but smaller-than-expected improvement in fertilization success. A pair of optimality models predict that, despite the large difference in the energetic value of egg contents and jelly material, the presence of the jelly coat does not diminish selection for larger egg cell size when sperm are limiting.     

Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

On the contents of the cortical granules from Xenopus laevis eggs

The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.     

Immunolocalization of the sea urchin sperm receptor in eggs and maturing ovaries

The sperm receptor from Strongylocentrotus purpuratus eggs is a high molecular weight proteoglycan-like molecule that inhibits fertilization species-specifically in a competition bioassay. A preparation of highly active sperm receptor that contained one major N-terminal sequence was used to generate polyclonal antibody in rabbits. This antibody species-specifically inhibited fertilization at low concentrations without interfering with fertilization envelope elevation. On immunofluorescence microscopy, the antibody recognized determinants on the mature egg cell surface and in the cortical granules just beneath the surface. Preabsorption of the antibody with the calcium-soluble fraction of the exudate from cortical granules rendered the antibody specific for the cell surface in mature eggs and still able to inhibit fertilization at the same concentrations as before treatment with cortical granule exudate. With antibody preabsorbed with cortical granule and by counting antibody-gold particles viewed by electron microscopy, sperm receptor was almost undetectable on the cell surface of immature oocytes in preseason ovaries, present on the cell surface and intracellularly in immature oocytes of ovaries collected at the beginning or at the height of the spawning season, and present only on the cell surface of mature oocytes in the lumen of the ovaries. Our results indicate that receptor is synthesized early in oogenesis and is rapidly moved to the egg cell surface.     

Xenopus laevis egg jelly contains small proteins that are essential to fertilization.

The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4 degrees C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight "structural" glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS-PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.     

Xenopus laevis Egg Jelly Contains Small Proteins That Are Essential to Fertilization

The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4°C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight “structural” glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS–PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.     

革胡子鲶受精过程的扫描电镜观察

应用扫描电镜观察和描述了革胡子鲶成熟卵和精子的形态、卵壳膜的表面结构和形态、受精孔的位置和结构、精子入卵过程的程序和变化。讨论了精子入卵过程及精孔细胞在解体之后可能转变为一种能够吸引精子在精孔区聚集的“受精素”物质等问题。     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

Glycolipid linkage of a polyspermy blocking glycosidase to the ascidian egg surface.

Ascidian eggs release N-acetylglucosaminidase rapidly into the seawater following fertilization. This glycosidase is detected seconds after fertilization, and histochemical tests suggest the cell surface as the prefertilization storage site (Lambert, C. C. (1989). Development 105, 415-420). Living eggs of Ascidia ceratodes, A. callosa, and A. paratropa all cleave a fluorogenic substrate in seawater. Following cell surface biotinylation and activation of the eggs, enzyme activity binds to streptavidin further substantiating the cell surface localization. The released glycosidase has a molecular weight of 180 kDa by size exclusion chromatography and exhibits bands at 62 and 70 kDa by SDS-PAGE, suggesting a possibly multimeric enzyme. The enzyme is released by a glycophosphatidylinositol-specific phospholipase C and HNO2 deamination, both of which are specific indicators of linkage to the cell surface via phosphatidylinositol. The enzyme from unfertilized eggs is quite hydrophobic in Triton X-114 phase partition experiments but becomes hydrophyllic after release by activation or deamination. All of these observations are consistent with the glycosidase being anchored to the cell surface via a GPI anchor that is cleaved at fertilization to yield the soluble form of the enzyme which helps protect the egg against polyspermy. We discuss the possible role of a cell surface PLC in this release.     

Changes in the topography of the sea urchin egg after fertilization

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.     

Surface ultrastructure of paddlefish eggs before and after fertilization

The surface ultrastructure of eggs of the paddlefish Polyodon spathula was investigated by scanning electron microscopy. Mature eggs of paddlefish possess four to 12 micropyles in the animal polar region. There are sperm entry sites in the egg surface under the micropyles which consist of tufts of microvilli. Five to nine sperm entry sites were observed on mature eggs. Probably, the number of sperm entry sites corresponds to the number of micropyles. In a few eggs, 1 min after fertilization the ball-like enlarged top of a cytoplasmic process (probably a full-grown fertilization cone) had reached the external aperture or the canal of several micropyles. In other micropyles of the same egg, a few smaller cytoplasmic processes or flocculent material were found in the micropylar canal. With one exception, no sperm tails were found there. The formation of the full-grown cytoplasmic process is possibly initiated before the cortical reaction has started in an area of the animal hemisphere. Three, 10 and 20 min after fertilization, the uneven surface of the cortical cytoplasm in the animal polar region rose gently where microvilli were much less than the in other area and together with a secondary polar body at the latter stage. Taken together, paddlefish eggs may have sperm entry sites corresponding to the number of micropyles and respond to the stimulus of fertilization by forming a few cytoplasmic processes–fertilization cones (larger and smaller). Sperm penetration into the egg may be achieved at an earlier stage of fertilization (sperm-egg contact), as inferred from the fact that a secondary polar body was formed at the 20-min stage irrespective of the exceptional finding of the sperm tail.     

An Electron Microscopic Study of Double Fertilization in Allohexaploid Wheat Triticum aestivum L.

Double fertilization has been examined by electron microscopyin allohexaploid wheat, Triticum aestivum L. cv. Mardler. Serialsections through fertilized ovules revealed that following dischargeof the pollen tube contents into the degenerate synergid thelatter extended to form a continuum between the egg and centralcells. The two naked sperm nuclei were seen at the far end ofthis extended synergid. These observations suggest that thedegenerate synergid may play a role in transporting the spermnuclei to the site where they can be accepted by the egg andcentral cell. In comparison with double fertilization in Plumbagozeylanica L., we also suggest that the degenerate synergid preventsmale cytoplasms from being transmitted to the egg and centralcell. The present study also confirms that in the fertilizedcentral cell the maternal and paternal genomes remain physicallyseparate at least until the first nuclear division of the coenocyticphase of endosperm development. Double fertilization, degenerate synergid, Triticum aestivum     

Limited proteolysis of some egg surface components is an early event following fertilization of the sea urchin, Strongylocentrotus purpuratus.

The surface proteins of eggs from Stronglocentrotus purpuratus were labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination. The eggs were examined after solubilization and disaggregation in sodium dodecyl sulphate (SDS) by electrophoresis on SDS-polyacrylamide slab-gels. Seventy-five percent of the label was found in material with a molecular weight greater than 130,000. About 5% of the radioactivity was excluded from the gels. Upon fertilization, embryos show a redistribution of the radioactively labeled species. There is a decrease in the amount of very high molecular weight material but an increase (35–40%) in material excluded from the gel. In addition, new radioactive bands of lower molecular weight are found. This change of distribution in the radioactive bands is blocked by inclusion of soybean trypsin inhibitor either before or immediately after fertilization, which completely inhibits the cortical granule protease. The disappearance of high molecular weight components is prevented by treatment of the eggs with procaine during fertilization, although the appearance of low molecular weight bands (approximately 20,000 and 30,000) is not completely blocked by procaine treatment. Parthenogenic activation of eggs by butyric acid or partial metabolic activation by ammonia each leads to changes in the egg surface proteins which are similar but not identical to those seen after fertilization. The data suggest that the labeling occurs on the vitelline membrane, plasma membrane and jelly layer. The possible significance of limited proteolysis in fertilization is discussed.     

INDUCTION OF THE ACROSOME REACTION ON THE EGG SURFACE OF DE-JELLIED SEA URCHIN EGGS BEFORE AND AFTER FERTILIZATION*

The capacity of the surface of sea urchin eggs to induce the acrosome reaction was assayed by estimating the rate of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs before and after fertilization. DTT-disruption of the vitelline coat did not eliminate the acrosome reaction-inducing capacity. This capacity was retained after fertilization in eggs of both H. pulcherrimus and A. crassispina. The acrosome reaction-inducing capacity of the eggs was markedly decreased by treatment with trypsin. The low capacity of the trypsin-treated eggs was maintained after fertilization in H. pulcherrimus, but in A. crassispina the capacity returned to the pre-trypsin treatment level after fertilization. Fertilized eggs from which the fertilization membrane was mechanically removed retained the inducing capacity to a considerable extent, independent of the presence or absence of the hyaline layer, but the capacity diminished rapidly as cleavage proceeded. It was concluded from these data that the acrosome reaction of spermatozoa actually occurred at the surface of de-jellied eggs and that the inducing substance resides in the plasma membrane in addition to the fertilization membrane. A chemical difference between the inducing substance of egg surface and jelly substance is discussed.     

Fine Structural Analysis of Animal Cell Surfaces: Membranes and Cell Surface Topography

Current research on the mechanism of transmembrane regulationof topographic modulation at the cell surface is described forthe sea urchin egg and Sarcoma 180 ascites tumor cells of SwissWebster white mice. The transmembrane system is characterizedin terms of three components: glycocalyx, membrane, and cytofibrillarstructures. The importance of membrane molecular architectureper se relative to the other surface components is assessedin terms of freeze fracture analysis of both cell types as wellas concanavalin A (ConA)-mediated long term agglutination, cytochalasinB effects, and other drug-induced changes at Sarcoma 180 cellsurfaces. A quantitative and qualitative assessment of intramembranousparticle (IMP) sizes and density distributions reveals intrinsicstructural changes of the fusing membranes at cortical reactionduring sea urchin egg fertilization and also with the post-fertilizedaccumulation of surface microvilli. Comparable changes in IMPare noted for microvillus retraction and membrane smoothingin Sarcoma 180 cells under a variety of experimental conditions.On the other hand, chemical perturbation of S-180 cell surfacesreveals a rather non-ubiquitous, though identifiable, involvementof microfilaments and no microtubule involvement in these topographicchanges. These observations suggest that the plasma membraneis a dynamic structure poised between "restrictive" and "lessrestrictive" states of fluidity or deformability and, hence,is a determinant component in topographic change.