Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.     

The cortical actin cytoskeleton of unactivated zebrafish eggs: Spatial organization and distribution of filamentous actin,nonfilamentous actin,and myosin-II

Actin and nonmuscle myosin heavy chain (myosin-II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS-polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase-I, or anti-actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin-II in whole mounts and egg fragments. Myosin-II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin-II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin-II provides insight into possible functions of these proteins before and after fertilization. © 1996 Wiley-Liss, Inc.     

The ultrastructure of the sea urchin egg cortex isolated before and after fertilization

The three-dimensional organization of cortices isolated from unfertilized and fertilized Strongylocentrotus purpuratus eggs has been examined by several techniques of light and electron microscopy. It has been found that when moderate shear forces are used, the isolated unfertilized egg cortex, in addition to cortical granules, contains acidic vesicles and an elaborate network of rough endoplasmic reticulum. This network provides a physical link between the cell surface and several kinds of cytoplasmic organelles (mitochondria, yolk granules, acidic vesicles) which are retained as part of the isolated cortex when gentle shear forces are applied. Furthermore a good visualization of actin in the cortex is provided: it is present as short filaments and mostly within the stubby microvilli of the egg. Finally, it has been noted that plaques exist on the inside face of the plasma membrane ready to assemble into typical clathrin coats that prefigure the burst of coated vesicle endocytosis that takes place after fertilization. The cortex isolated soon after fertilization is shown to contain coated pits and a scaffolding of filaments (mostly actin) in which many acidic vesicles are embedded.     

pH regulates the polymerization of actin in the sea urchin egg cortex

The state of actin in the isolated cortex of the unfertilized sea urchin egg can be controlled by experimentally manipulating the pH of the isolation medium. Cortices isolated at the pH of the unfertilized egg (6.5--6.7) do not contain filamentous actin, while those isolated at the pH of the fertilized egg (7.3--7.5) develop large numbers of microvilli which contain bundles of actin filaments. Cortices that are isolated at pH 6.5 and then transferred to isolation medium buffered at pH 7.5 also develop actin filaments. However, the filaments are not arranged in bundles and microvilli do not form. Although the cortical granules in cortices isolated at pH 6.5 discharge at a free Ca++ concentration of approximately 10 micrometer, actin polymerization is not induced by increasing the Ca++ concentration of the isolation medium. These results suggest that the increase in cytoplasmic pH which occurs following fertilization induces the polymerization of actin in the egg cortex.     

Influence of the polyamine spermine on the organization of cortical filaments in isolated cortices of Xenopus laevis eggs

Microinjection of spermine into Xenopus laevis eggs induces precocious furrowing, and together with known spermine-actin interactions, suggests that spermine may be affecting cortical microfilaments involved in cytokinesis. An electron microscopic study of injected eggs revealed that the ultrastructure of the induced furrows was similar to that of both artificially activated eggs and fertilized eggs. In isolated egg cortices, increasing spermine concentrations (1, 3 and 10 mM) resulted in marked changes in cortical microfilament organization. At low concentrations, spermine appeared to stabilize microfilaments and at higher concentrations induced lateral associations between filaments and formation of bundles. The actin nature of these cortical microfilaments was confirmed by immunocytochemistry. The electrophoretic profiles of proteins from control and spermine-treated isolated cortices were similar. Although the total protein content of isolates in 3 and 10 mM spermine was elevated, the relative actin content remained constant. The results are in agreement with previous in vitro studies of polyamine interactions with actin and support the hypothesis that a polyamine-actin interaction may be important in the regulation of cytokinesis.     

OCCURRENCE AND FINE STRUCTURE OF SUBCORTICAL CYTOPLASMIC ISLETS IN FERTILIZED EGGS OF CYNOPS PYRRHOGASTER

Fertilized and unfertilized eggs of Cynops pyrrhogaster were examined by light and electron microscopy. In fertilized eggs that have just been laid, there are numerous small cytoplasmic patches free of granules in the pigmented layer of the animal hemisphere. Many of these granule-free cytoplasmic islets gradually grow out subcortically from the pigmented layer and fuse to form a subcortical layer of yolk-free cytoplasm of varying thickness by the time of the first cleavage division. The cytoplasmic islets are present in 100% of the fertilized eggs, but not in unfertilized eggs. Electron microscopic observations showed that the cytoplasmic islets contain tubules and that development of a complex system of cortical tubules constitutes the basis of the early growth of the cytoplasmic islets. The cortical tubules are transient structures and are no longer observable a few hours after the eggs are laid. These phenomena are considered to be a response of the egg to the fertilization stimulus.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

Alpha-actinin from sea urchin eggs: biochemical properties, interaction with actin, and distribution in the cell during fertilization and cleavage

A protein similar to alpha-actinin has been isolated from unfertilized sea urchin eggs. This protein co-precipitated with actin from an egg extract as actin bundles. Its apparent molecular weight was estimated to be approximately 95,000 on an SDS gel: it co-migrated with skeletal-muscle alpha-actinin. This protein also co-eluted with skeletal muscle alpha-actinin from a gel filtration column giving a Stokes radius of 7.7 nm, and its amino acid composition was very similar to that of alpha-actinins. It reacted weakly but significantly with antibodies against chicken skeletal muscle alpha-actinin. We designated this protein as sea urchin egg alpha-actinin. The appearance of sea urchin egg alpha-actinin as revealed by electron microscopy using the low-angle rotary shadowing technique was also similar to that of skeletal muscle alpha-actinin. This protein was able to cross-link actin filaments side by side to form large bundles. The action of sea urchin egg alpha-actinin on the actin filaments was studied by viscometry at a low-shear rate. It gelled the F-actin solution at a molar ratio to actin of more than 1:20, at pH 6-7.5, and at Ca ion concentration less than 1 microM. The effect was abolished by the presence of tropomyosin. Distribution of this protein in the egg during fertilization and cleavage was investigated by means of microinjection of the rhodamine-labeled protein in the living eggs. This protein showed a uniform distribution in the cytoplasm in the unfertilized eggs. Upon fertilization, however, it was concentrated in the cell cortex, including the fertilization cone. At cleavage, it seemed to be concentrated in the cleavage furrow region.     

The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.     

Morphological features of the surface of the sea urchin (Arbacia punctulata) egg: Oolemma-cortical granule association

Scanning microscopy and transmission electron microscopy of sectioned specimens and freeze-fracture replicas revealed the presence of slightly elevated regions, approximately one-fourth to one-half the diameter of microvilli, which were situated along the surface of unfertilized Arbacia eggs. These modifications of the surface of the egg were observed in areas occupied by cortical granules and were greatly reduced in number following the cortical granule reaction. Few such modifications were present in immature and urethane-treated ova, in which cortical granules were located in regions of the egg other than the cortex. Freeze-fracture replicas of unfertilized eggs revealed a significantly higher density of intramembranous particles within the plasmalemma when compared to replicas of the membrane surrounding cortical granules. Areas characteristic of the cortical granule membrane, i.e., sparsely laden with particles, were not observed within the plasmalemma of the fertilized egg. Hence, following its fusion with the egg plasma membrane there is a dramatic reorganization in particle distribution of the membrane derived from cortical granules.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.     

Cytoskeleton of the unfertilized sea urchin egg

Unfertilized Paracentrotus lividus egg cytoskeleton is prepared by mild, nonionic detergent extraction at 4 degrees C in buffer systems containing either 2-methyl-2,4-pentanediol (hexylene glycol) or glycerol. These extractions allow the isolation of cytomatrices that maintain the egg form and are 70-80 micron in diameter. DNase inhibition assays show that actin is in polymerized form in these cytomatrices. Ultrastructural observations reveal that the cytoskeletons are made up essentially of 2 categories of filaments, 7-8-nm and 2-4-nm in diameter, respectively. After heavy meromyosin labelling, short, radiating actin filaments are seen in the cortical region, while longer actin filaments are found in the internal region of these cytomatrices. The 2-4-nm filaments of still unknown biochemical nature are organized in a meshwork. In contrast to results found with fertilized eggs, bundles of actin filaments and microtubules are absent; 8-13-nm filaments are not detected.     

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.     

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.     

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.     

The distribution of polymerized actin in the rat egg and its sensitivity to cytochalasin B during fertilization

The distribution of polymerized actin in rat eggs fertilized in vitro was determined using NBD-phallacidin (NBD-ph). Unfertilized and fertilized eggs exhibited a 3-5-micron-thick band of fluorescence that encompassed the entire cortical cytoplasm. There was no dramatic increase in the staining of the cortex in association with any component of the fertilizing sperm during its incorporation into the egg. Unfertilized eggs and fertilized eggs obtained at intervals after sperm-egg fusion were treated with cytochalasin B (CB; 5 micrograms/ml) and subsequently stained with NBD-ph. Unfertilized eggs treated with CB exhibited a continuous ring of cortical staining identical to that seen in untreated eggs. Eggs treated with CB 15 min after sperm-egg fusion exhibited small gaps in the cortical staining pattern, whereas those exposed to CB 1 hr after fusion exhibited larger gaps and the staining pattern appeared punctate. This pattern could be seen throughout the remainder of the 7 hr period of sperm incorporation and for at least 13 hr thereafter. CB-treated fertilized eggs that were washed to remove the drug again exhibited uninterrupted cortical staining on treatment with NBD-ph. CB also induced the resorption of surface elevations that are normally seen on the eggs during sperm incorporation, but it did not affect the morphology of unfertilized eggs. The sensitivity to CB during fertilization coincides with the onset of a variety of egg shape changes that occur during the period of sperm incorporation (Battaglia and Gaddum-Rosse, Gamete Res., 10:107-118, 1984a).(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrins on eggs: the betaC subunit is essential for formation of the cortical actin cytoskeleton in sea urchin eggs

Eggs of several metazoans have been demonstrated to express integrins; however, their function is unclear. Previous studies have shown that the betaC integrin subunit is expressed on unfertilized sea urchin eggs and proteolytically removed at fertilization. Here we report that the betaC subunit is reexpressed on the egg surface immediately after fertilization. Using morpholino antisense oligonucleotides to block translation, we show that without betaC expression, eggs undergo cleavage resulting in loosely adherent cells that fail to develop beyond a blastula. Without betaC containing integrins, the cortical actin network of the egg does not form, yet contractile rings appear. Coinjection of RNA encoding the betaC or chicken beta1 subunit, but lacking the morpholino target sequence, rescues the cortical actin network and normal embryos result. Coinjection of RNA encoding the betaC subunit lacking the cytoplasmic domain fails to rescue. These studies demonstrate that the cortical actin cytoskeleton is anchored by betaC integrins and contractile ring actin is not. We suggest that one important function of egg integrins is to organize the actin cortex.     

Preparation of plasma membranes from fertilized sea urchin eggs

A new method is presented for preparation of highly purified plasma membranes from fertilized sea urchin eggs. The purified plasma membranes are in vesicle form and are highly enriched in ouabain inhibitable, Na+/K+ ATPase activity. Analysis of membrane proteins by sodium dodecyl sulfate-gel electrophoresis indicates that several high-molecular-weight proteins characteristic of plasma membranes from unfertilized eggs are absent in plasma membranes from fertilized eggs.     

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.