Purification and properties of soluble actin from sea urchin eggs

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.     

Na movements and their oscillations during fertilization and the cell cycle in sea urchin eggs

We have analysed in detail the Na⁺ content and Na⁺ influx during fertilization and first divisions of the sea urchin egg (Paracentrotus lividus) using a filtration technique devised to eliminate rapidly contamination by the Na⁺ of external sea water. In the first 5 min following fertilization the egg fills up with Na⁺ (+ 30%). Thereafter Na⁺ is extruded and the Na⁺ content stabilizes at about 60% of the unfertilized egg level by the second cleavage (2 h). The initial increase in Na⁺ content is due to a large increase in Na⁺ influx already detected at 20 sec. The Na⁺ influx reaches its maximum at 1 min and its minimum at 5 min. H⁺ excretion follows the same kinetics. A second increase in Na⁺ influx is noted 5–10 min after fertilization; it reaches its maximum at prophase metaphase (30 min) and its minimum during cleavage (60 min). These oscillations in Na⁺ influx were observed for the first three divisions. Fertilization also immediately stimulates the Na⁺ efflux which remains elevated throughout the cell cycle and is responsible for the depletion of the Na⁺ content of the embryos. Activation of the eggs by weak amine bases (5 mM NH₄Cl) which bypasses the early cortical reaction produces only a depletion in the Na⁺ content of the egg similar to that produced by fertilization. NH₄Cl also increases the Na⁺ influx soon after fertilization, although no transient variations are noted.     

Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs.

A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage

1. 1. Changes in stiffness of the cell surface at fertilization and during cleavage in sea urchin eggs were determined by the magnetic particle method.

2. 2. The stiffness of the cell surface increased at fertilization, reached a maximum after about 1.5 min, then decreased and reached a minimum about 4 min after insemination, followed by a gradual increase, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5 °C.

3. 3. The stiffness of the cell surface increased during the diaster stage, reached a maximum 2 to 3 min before the onset of cleavage, then decreased to a minimum about 1 min before the onset of cleavage, increased again, reached a maximum during cleavage and then diminished, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5 °C. A similar stiffness change was observed in the eggs of Hemicentrotus pulcherrimus at 17 to 19 °C, occurring almost in parallel in both the equatorial and polar surfaces.

     

Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM- or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC- or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 microM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.     

Effects of Bonellin on fertilization and cleavage of the eggs of the sea urchin,sphaerechinus granularis

We have studied the structural changes occurring in the eggs of the sea urchin Sphaerechinus granularis treated with bonellin, the green pigment of the sea worm Bonellia viridis, which is responsible for the masculinization of the larva of this animal. The two major targets of bonellin appear to be the cortical structures and the nuclear membrane, while the mitochondria do not appear to be affected. As a result of bonellin treatment, cleavage is prevented while nuclear divisions proceed. The possibility is discussed that the alteration of the cortical structures may interfere with the assembly of the surface microfilaments and hence with the formation of the cleavage furrows.     

Actin, microvilli, and the fertilization cone of sea urchin eggs

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.     

The NO pathway acts late during the fertilization response in sea urchin eggs

Both the inositol 1,4,5-trisphosphate (InsP(3)) and ryanodine receptor pathways contribute to the Ca(2+) transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP(3) receptor pathway has a primary role in causing Ca(2+) release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca(2+) at fertilization and establish that NO levels rise after, not before, the Ca(2+) wave is initiated and that this rise is Ca(2+)-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca(2+) transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca(2+) wave to regulate the duration of the fertilization Ca(2+) transient.     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

Distribution of fluorescently labeled actin in living sea urchin eggs during early development

Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.     

Source and sinks for the calcium released during fertilization of single sea urchin eggs

The source and sinks for the intracellular calcium released during fertilization were examined in single eggs from the sea urchin, Arbacia punctulata. Single eggs were microinjected with the calcium photoprotein, aequorin. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or observed with a microscope-image intensifier-video system. In the normal egg a propagated release has been observed. The source of the calcium was investigated in the organelle-stratified centrifuged egg and by the use of mitochondrial uncouplers. In the organelle-stratified centrifuged egg, the calcium-aequorin luminescence was found to originate from the clear zone. The principal constituent of the clear zone is the endoplasmic reticulum. Other potential sources of calcium are the mitochondria. Their contribution to the calcium transient was investigated by exposure of aequorin-injected eggs to mitochondrial uncouplers either before or after fertilization. There was no calcium released from the mitochondria before fertilization. A very large calcium store was released from the mitochondria after fertilization. Interestingly, eggs fertilized in the presence of uncouplers showed no increase in the calcium-aequorin luminescence over untreated eggs. Apparently, in the absence of mitochondrial uptake, other sinks for calcium with affinity and capacity similar to the mitochondria exist, but their nature is unknown. We suggest that the endoplasmic reticulum is the source of the intracellular calcium released upon fertilization and that the mitochondria are the principal sink. The results are discussed with regard to the metabolic activation of the egg.     

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.     

Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at approximately 23 s as measured by calcium- aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at approximately 51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.     

Histones in the first cleavage cycle of fertilised sea urchin eggs

Nuclei were isolated from Echinus eggs through the first cleavage cycle by modification of existing techniques. When these nuclei were extracted with 2 M NaCl and the supernatant diluted to 0.15 M, large amounts of non-histone proteins remained in solution. The precipitated nucleoprotein contained expected amounts of DNA and a protein analogous to mammalian histone H1. Extrachromosomal histone H1 was eliminated by the modified isolation procedure. Amounts of nuclear proteins soluble in 0.15 M NaCl reached a peak in G2. Histones and non-histone proteins were phosphorylated postfertilization, in early prophase and in telophase.     

Cold shock induces actin reorganization and polyspermy in sea urchin eggs

The effect of low temperature on the cortical actin system and polyspermy in sea urchin eggs was studied. Eggs cold-shocked at 3 degrees C for 1 hour formed a cortical system of polymerized actin and were polyspermic when fertilized. These effects were completely reversible following a 7-20 minute recovery period at room temperature. The present data raises the possibility that actin, or components of the egg cytoskeleton, regulate sperm entry in sea urchin eggs.     

Changes in the potassium content of sea urchin eggs on fertilization

In the eggs of Arbacia lixula and Paracentrotus lividus an uptake of K occurs during the first 10 minutes following fertilization. Between 10 and 40 minutes K is then released. Both in Arbacia and in Paracentrotus the minimum point of the curve coincides with the nuclear streak stage. A maximum loss of 25 per cent in Arbacia and 20 per cent in Paracentrotus with respect to the amount present in the unfertilized eggs has been found. From 40 minutes up to 1 hour K undergoes a further increase and when the first cleavage sets in the same amount of K is present as in the unfertilized eggs. By treating the eggs with K-free artificial sea water it has been established that about 60 per cent of the K content of the eggs is in a non-diffusible condition. Also under such conditions the eggs when fertilized are able to take up even the very small amount of K present in the medium that was released by them prior to fertilization.