Partially Fertilized Sea Urchin Eggs: An Electrophysiological and Morphological Study.

Propagation of the cortical reaction in sea urchin eggs may be interrupted by a mild heat shock. In such partially fertilized eggs three distinct cortical zones may be distinguished. First, an activated area where cortical granule exocytosis is complete, the fertilization membrane is elevated, and there is a cortical meshwork of polymerized actin. Second, at the antipode an area where the cortical granules are intact, actin is not polymerized, and the surface structure in general resembles that of the virgin egg. Between the two there is a transitional zone, some 10 to 20 μm wide, where a fraction of cortical granules have exocytosed, giving rise to isolated "blebs" of elevated fertilization membrane. Partially fertilized eggs have resting potentials ranging from −20 to −80 mV, and upon re-insemination give rise to step depolarizations indicating that spermatozoa may interact and possibly fuse with the "unactivated surface".     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jelly-treated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT- or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Bioelectric responses of sea urchin eggs inseminated with oyster spermatozoa: a sperm evoked potential without egg activation

Multiple oyster spermatozoa can enter sea urchin eggs with or often without fertilization membrane formation (Osanai and Kyozuka, 1982). In the present work, electrical responses of sea urchin (Temnopleurus hardwicki) eggs inseminated with oyster (Crassostrea gigas) sperm were examined and correlated to the failure of monospermy and egg activation. With diluted sperm, a transient depolarization of the membrane with a constant pattern appeared repeatedly and discretely, and the depolarizations (sperm evoked potentials, SEPs) were not associated with fertilization membrane elevation. With dense sperm, the SEPs occurred consecutively, and sometimes an assembled consecutive depolarization was followed by an activation potential associated with cortical granule discharge. When the membrane potential was artificially held at positive levels, the frequency of SEPs was strongly suppressed but not completely blocked. The present results indicate that an individual heterologous spermatozoon neither produces a depolarization sufficient to block additional sperm entry, nor stimulates egg activation, and that simultaneous entries of multiple heterologous spermatozoa, as possibly reflected by the assembled consecutive depolarizations, induce cortical granule discharge and egg activation.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Maturation and fertilization of the sea urchin oocyte: An electrophysiological study

Some electrical properties of the sea urchin oocyte during germinal vesicle breakdown (GVBD) and fertilization have been studied using two intracellular electrodes. Oocytes with distinct germinal vesicles have resting potentials of ?70 to ?90 mV and the specific membrane resistance may range from 3 to 10 kΩ·cm². Around rest the I–V relationship is concave toward the axis origin and the membrane is K⁺ selective. A second electrical state, of lower potential and higher resistance, preexists in the membrane. Following GVBD, the K⁺-selective system is lost and the oocyte attains the characteristics of the second state with a resting potential of ?10 to ?50 mV and specific membrane resistance of 10–50 kΩ·cm². At rest the I–V relationship tends to be convex toward the axis origin. The majority of sea urchin eggs (which have undergone GVBD and completed meiosis) have a resting state of ?10 to ?30 mV; 10–50 kΩ·cm². The I–V relationship around rest is convex toward the axis origin and the resting potential is sensitive to changes of Na⁺, Cl^?, and K⁺ in the external medium. There is probably no major change in the electrical properties of the oocyte during the completion of meiosis. A small percentage of eggs from suboptimal animals have high resting potentials of ?70 to ?90 mV and specific membrane resistance of 5–50 kΩ·cm². Such eggs have predominantly K⁺-selective membranes and we suggest that they are either underripe or aged. The first electrical event across the egg plasma membrane during fertilization is a step-like depolarization which occurs about 2 sec after the attachment of the fertilizing spermatozoon to the vitelline layer. There is no change—at the level of the light microscope—either in the egg surface or in the behavior of the spermatozoon until the second event, the fertilization potential (FP), is initiated 11 sec later. The cortical reaction occurs simultaneously with the FP and during the rising phase of the FP the spermatozoon stops gyrating around its point of attachment. Oocytes, which do not have cortical granules, upon insemination exhibit step events but no FP; in contrast artificially activated eggs, either spontaneous or induced by the ionophore A23187, give rise to only the FP. We suggest that the FP is the electrical result of the modification of the egg plasma membrane during cortical exocytosis.     

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. II. FERTILIZATION IN ACIDIFIED SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA*

Fertilization of sea urchin eggs fails to occur at a pH lower than 6.5. Analytical studies on this problem were made with Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus. If the spermatozoa have been pretreated with egg water, eggs can be fertilized even at pH 6.5 and 6.0. The acrosome reaction is inhibited at a pH lower than 6.5. Intact spermatozoa fail to adhere to the fixed eggs in acidified sea water, whereas egg-water-treated spermatozoa adhere even at pH 6.5 and 6.0. From these results we infer that the failure of fertilization at pH 6.5–6.0 is caused by non-occurrence of the acrosome reaction, and that fertilization reactions other than the acrosome reaction, such as the binding and fusion of the gametes, are not inhibited in this range of pH. At pH 5.5, the spermatozoa become inert and fertilization is inhibited or suppressed, even though egg-water-treated spermatozoa are employed.     

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.     

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors

Protease inhibitors were used to study certain physiological responses (secretion of the cortical granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was completely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to prevent subsequently added sperm from penetrating (fertilizing) ionophore-activated eggs, co-incident with the elevation of the fertilization membrane. These processes were retarded by inhibitors of the cortical granule protease in ionophore-activated eggs, just as they are when eggs are initially stimulated by sperm at fertilization. A23187-activated eggs did not divide unless they had been secondarily fertilized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles required to organize the mitotic apparatus. The centrioles are normally provided by the sperm during fertilization. A23187 may also be toxic to the eggs. Furthermore, since cortical granules are secretory organelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.     

Rho, Rho-kinase, and the actin cytoskeleton regulate the Na+ -H+ exchanger in sea urchin eggs

At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.     

Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization

Sulfated acid mucopolysaccharides have been shown to be constituents of cortical granules in sea urchin and vertebrate eggs. These observations were made possible by retaining soluble acid mucopolysaccharides in situ within the eggs by precipitation during fixation with cetyltrimethyl-ammonium bromide, a quaternary ammonium salt. The sulfated mucopolysaccharides were then identified by staining with Astrablau at pH 0.2 and also by reaction with sodium rhodizonate. Staining reaction with Alcian blue at pH 2.5 showed that carboxylated mucopolysaccharides may also be present in cortical granules. The natural ionic environment of these eggs would favor the formation of very stable complexes between sulfated mucopolysaccharides and quaternary ammonium salts. Brief exposure of unfertilized sea urchin eggs to several quaternary ammonium compounds produced a residual adverse effect on subsequent fertilization in terms of increased vulnerability to polyspermy and reduced fertilizability. These results suggest that sulfated acid mucopolysaccharides participate in the function of the cortical granules and the establishment of the block to polyspermy at fertilization, and possibly in other cellular secretory processes.     

ELECTRICAL POLYSPERMY BLOCK IN SEA URCHINS: NICOTINE AND LOW SODIUM EXPERIMENTS1

Nicotine reduces the amplitude of the fertilization potential in sea urchin eggs, at least in part because it decreases the slope of the current voltage relation of the unfertilized egg membrane. The reduced fertilization potential amplitude provides an electrophysiological explanation for previous observations that nicotine impairs the fast block to polyspermy. The block to polyspermy is also impaired by fertilization in low sodium sea water, a medium which has been reported to reduce fertilization potential amplitude.     

Fertilization of the starfish Astropecten aurantiacus

In the starfish Astropecten aurantiacus the acrosome reaction occurs when the spermatozoon contacts the outer surface of the jelly layer. A long thin acrosomal filament is extruded from the anterior region of the spermatozoon and establishes contact with the oocyte surface. This latter interaction initiates the movement of the spermatozoon to the oocyte surface, formation of the fertilization cone and the cortical reaction. The first detectable electrical change across the oocyte plasma membrane during interaction with the spermatozoon is the fertilization potential (FP) which occurs simultaneously with the cortical reaction. The FP is probably the electrical result of the modification of the oocyte plasma membrane during cortical exocytosis. There are no primary step-like depolarizations during fertilization of starfish oocytes, which contrasts with the situation in sea urchin eggs [see 13]. We suggest that the difference in electrical response to fertilization of starfish oocytes and sea urchin eggs may be attributed to the location of the acrosome reaction in these animals and not to their different meiotic states.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.     

Non-propagated cortical reactions induced by the divalent ionophore A23187 in eggs of the sea urchin, Lytechinus variegatus

The divalent ionophore A23187 can induce a non-propagated discharge of cortical granules in sea urchin eggs resulting in the elevation of partial fertilization membranes. This occurs when unfertilized eggs (1) partially overlap the margin of a solid film of the ionophore; (2) touch a Sephadex bead previously infiltrated with the ionophore; or (3) are exposed to solutions of ionophore for short periods (15–30 sec). These results demonstrate that the release of cortical granules does not automatically induce the discharge of neighboring granules.     

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.