Unfertilized Sea Urchin Eggs Research Articles

The use of caged substrates to assess the activity of 6-phosphogluconate dehydrogenase in living sea urchin eggs

As part of our inquiries into the regulation of the hexose monophosphate shunt in the early development of sea urchin eggs and embryos, we have developed a novel method to assess the in vivo activity of the enzyme 6-phosphogluconate dehydrogenase (6PGDH) before and after fertilization. Our measurements show that the intracellular level of 6-phosphogluconate (6PG) in eggs decreases 60% after fertilization, which is consistent with the increase in the activity of 6PGDH previously reported using irreversibly permeabilized cell assays (Swezey and Epel, Proc. Natl. Acad. Sci USA 85, 812–816, 1988). The in vivo turnover of the 6PG pool was assessed using a new radioisotopic technique. 1- 14C-labeled 6PG was chemically modified such that it was not metabolized by cellular 6PGDH and could be rapidly converted back to 6PG by photolysis. This “caged” 6PG was introduced into unfertilized sea urchin eggs using a transient permeabilization procedure, and then the oxidation of [1- 14C]6PG in vivo upon irradiation was followed. Oxidation of 6PG was complete within 7–11 s of irradiation, indicating an extremely rapid turnover of this pool in sea urchin eggs. Based on the 6PG pool sizes and the kinetic properties of 6PGDH, determined here, along with the activity levels seen in permeabilized cells, the half-time for the label in the 6PG pool in sea urchin eggs is calculated to be 26 s. This is inconsistent with the in vivo turnover rates seen in these studies, indicating that the permeabilized cell assays overestimate the degree of inhibition of 6PGDH before fertilization. These results suggest that caution should be exercised in extrapolating data obtained from permeabilized cells to the situation in vivo.

The kinetics of autolysis in osmotically stressed sea urchin eggs

We have measured the kinetics of death from hyperosmotic stress in unfertilized sea urchin eggs in an attempt to dissect the two conflicting influences of temperature lowering below the freezing point: as more ice forms during temperature lowering, the rate of injury increases rather than decreases. Two models are examined. An Arrhenius model, in which the osmotic stress imposed by extracellular ice formation is envisaged as reducing the “activation energy”, provides a qualitative model but fails to fit the data in detail. In contrast, a Johnson—Mehl—Avrami nucleation model, in which the osmotic stress triggers an autolysis that is essentially independent of temperature or degree of osmotic stress above a threshold value, fits the data well. Two separate decay processes were seen, for which the time constants were 269s and 658s and the Avrami exponents were 7 and 1 respectively. The latter is an ordinary first-order attrition, though its independence from temperature distinguishes it from an Arrhenius model. The nucleation model is difficult to furnish with a simple physical representation.

Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.

Two distinct isoforms of sea urchin egg dynein.

Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-2 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.

Zinc increases the longevity of unfertilized sea urchin eggs

We have found that Zn2+ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn2+ inhibited this proteolysis. Therefore, Zn2+ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.

Fertilization‐Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self‐assembly of an extracellular matrix

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self-assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick-frozen, deep-etched, and rotary-shadowed eggs, are illustrated.

Guanosine 5'-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm.

We show that microinjecting guanosine-5'-thiotriphosphate (GTP gamma S) into unfertilized sea urchin eggs generates an intracellular free calcium concentration [( Ca]i) transient apparently identical in magnitude and duration to the calcium transient that activates the egg at fertilization. The GTP gamma S-induced transient is blocked by prior microinjection of the inositol trisphosphate (InsP3) antagonist heparin. GTP gamma S injection also causes stimulation of the egg's Na+/H+ antiporter via protein kinase C, even in the absence of a [Ca]i increase. These data suggest that GTP gamma S acts by stimulating the calcium-independent production of the phosphoinositide messengers InsP3 and diacylglycerol (DAG). However, the fertilization [Ca]i transient is not affected by heparin, nor can the sperm cause calcium-independent stimulation of protein kinase C. It seems that the bulk of InsP3 and DAG production at fertilization is triggered by the [Ca]i transient, not by the sperm itself. GDP beta S, a G-protein antagonist, does not affect the fertilization [Ca]i transient. Our findings do not support the idea that signal transduction at fertilization operates via a G-protein linked directly to a plasma membrane sperm receptor.

Reinvestigation of DNA ligase I in axolotl andPleurodelesdevelopment

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.

Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP.

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.

A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg

Multiple second messenger pathways have been proposed for transduction of the sperm-egg fusion event during fertilization of sea urchin eggs. Cytoplasmic alkalinization due to increased Na +H + antiport has been causally linked to many of the metabolic events during fertilization. Two possible second messenger pathways coupling sperm-egg fusion and antiporter activity are activation of protein kinase C (PKC) and Ca 2+-calmodulin kinase. A selective inhibitor of PKC is PKC(19–36), a synthetic peptide of the pseudosubstrate domain of the kinase. Injection of PKC(19–36) into unfertilized sea urchin eggs blocked cytoplasmic alkalinization during activation by phorbol 12-myristate 13-acetate, a PKC agonist. The rise in pH during fertilization was partially blocked by PKC(19–36), which suggested that multiple pathways regulate the antiporter during fertilization. The use of fluorescein chromophores to measure intracellular pH in sea urchin eggs is also discussed.

Isolation, characterization, and immunochemical properties of a giant protein from sea urchin egg cytomatrix

A giant protein of apparent molecular weight ( M r) 2000 kDa, as determined by SDS-PAGE, was isolated and partially purified, under denaturing conditions, from the detergentresistant cytomatrix of unfertilized sea urchin egg. Immunoblot analysis and indirect immunofluorescence microscopy observations indicated that this highmolecularweight protein crossreacted with the immunospecific serum raised against chicken breast muscle β-connectin. However, rotaryshadowing electron microscopy images of the protein revealed short threadlike structures which appear morphologically different from β-connectin structure. Indirect immunofluorescence localization of the protein with antiβ-connectin serum showed a distribution throughout the whole unfertilized egg cytomatrix. This immunofluorescence pattern seems to change upon egg fertilization, since at metaphase the fluorescence stain appears to be excluded from the mitotic apparatus region as revealed by the double immunolabeling with anti-β-connectin serum and monoclonal anti-α-tubulin antibody. Moreover, when egg cortical fragments were doublelabeled with anti-β-connectin serum and rhodamin-conjugated phalloidin, it was observed that the microfilaments assembled after fertilization seem to be in close association with the protein at the cleavage furrow and other locations. The possible significance of this sea urchin egg connectin(titin)-like protein is discussed.

Stable, resealable pores formed in sea urchin eggs by electric discharge (electroporation) permit substrate loading for assay of enzymes in vivo.

We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.

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

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.

Analysis of supranucleosome particles from unfertilized eggs of sea urchins

Two discrete supranucleosomal particles that differ in their electrophoretic migration on 1% agarose gels were isolated from unfertilized sea urchin eggs. Both particles contain the same complement of cleavage stage (CS) chromosomal proteins, which is identical to the complete set of basic proteins isolated directly from chromatin by extraction with 0.25 N HCl. DNA fragments between 210 and 1500 bp were found in both particles, and the basic unit of DNA repeat length determined by micrococcal nuclease digestion was 126 ± 3 bp. The isolated nucleoparticles are electrophoretically stable over a wide range of DNA sizes (126–1500 bp) indicating that their structure is maintained by internal interactions among the CS chromosomal proteins, previously designated CS A through CS G. Based on these results we conclude that the CS chromosomal proteins are functionally equivalent to classical histones in their ability to direct higher ordered structures of chromatin.

Tubulin polymerization in unfertilized sea-urchin eggs induced by elevated temperature.

Spontaneous polymerization of tubulin was induced in unfertilized eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus by warming to temperatures 10-12 deg. C above the normal environment for each species. Indirect immunofluorescence microscopy showed that over a period of several hours polymerization proceeded from a fine crystal-like precipitate to larger and fewer higher-order structures. These structures differed morphologically between the two species: L. pictus formed cytasters, while S. purpuratus most often formed flame-like arrays. Nuclear cycles were not initiated, permitting long-term observation of steady-state polymer redistribution. After several hours only a few very large cytasters remained near the surface of L. pictus eggs and these tended to constrict to form protrusions or occasionally a cleavage furrow that divided the egg into two fragments. Staining with rhodamine-labeled phalloidin showed that actin was also concentrated in the cytasters, primarily at the periphery of the centrosphere-like regions. L. pictus cytasters could be recycled by repeated cooling and rewarming, and showed an accelerated rate of polymerization with each successive cycle. Cytasters could also be isolated as long as the initial procedures were carried out at the elevated temperature. Washed preparations were stable at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin.

In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called '50K-A' was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.

Quantitation of the dynein pool in unfertilized sea urchin eggs

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.

The localization of PI and PIP kinase activities in the sea urchin egg and their modulation following fertilization

Phosphatidylinositol phosphate (PIP) kinase activity is localized to the cortical region of unfertilized sea urchin eggs, while phosphatidylinositol (PI) kinase activity is found in both cortical and noncortical membranes. Following fertilization PIP kinase activity decreases, while PI kinase activity remains unchanged. The selective loss of PIP kinase activity is related to cortical granule exocytosis since the drop in activity does not occur if exocytosis is prevented by high hydrostatic pressure. When isolated cortices are exposed to elevated concentrations of calcium, both the PI and PIP kinase activities increase, suggesting that activation of these enzymes might occur when calcium levels increase within the fertilized egg prior to cortical granule exocytosis. The polyamine spermine also stimulates the formation of phosphatidylinositol bisphosphate at physiological concentrations.

Filamentous actin organization in the unfertilized sea urchin egg cortex

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2–3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.