Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

A cortical granule-specific enzyme,B-1,3-glucanase,in sea urchin eggs

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.     

Intermolecular cross-linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross-links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme-linked immunosorbent assay. VEs isolated by means of three published procedures cross-reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross-reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross-react with antiserum against FE ghosts. To determine which VE polypeptides were cross-linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross-reacted with the antibody.     

Properties of adenylate cyclase activity during early sea urchin development

The total adenylate cyclase activity in homogenates of eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus was assayed in vitro and found to remain constant in eggs before and at intervals after fertilization. In S. purpuratus egg homogenates virtually all of the enzyme activity was sedimented by centrifugation at 20 000 g. The enzyme specific activity in the 20 000 g pellet remained unchanged at each point through first cleavage, though it was several-fold higher than in the whole homogenate. The adenylate cyclase from both fertilized and unfertilized eggs was maximally active in vitro when assayed with 10 mM MgSO₄ and 10 mM NaF at pH 8 using 0.2 mM AMP-PNP (an ATP analog) as the substrate. Sucrose density gradient centrifugation of egg homogenates showed that adenylate cyclase activity was present in fractions which sedimented at a variety of densities. The adenylate cyclase specific activity in cortices isolated by the method of Sakai [10] from eggs at first cleavage was 4- to 6-fold higher than in unfertilized egg cortices. The increased enzyme activity in egg cortices at first cleavage suggests that adenylate cyclase-containing membranes may become localized within the egg cortex after fertilization.     

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.     

The glutathione thiol-disulfide status in the sea urchin egg during fertilization and the first cell division cycle

The intracellular levels of GSH, GSSG, and protein-glutathione disulfide (protein-SSG) have been measured in the eggs and developing embryos of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus. Total cellular glutathione is maintained in a very highly reduced state during these initial stages of development. Thus for unfertilized eggs of L. pictus the results (μmol/g dry weight) were 11 ± 1 for GSH, 0.02 ± 0.01 for GSSG, and 0.07 ± 0.02 for protein-SSG. No significant change in these values was observed upon fertilization of the eggs or during the first cell division cycle. The values obtained with S. purpuratus were somewhat greater, but were also found to exhibit no significant variations upon fertilization or cell division. These observations indicates that changes in the total cellular glutathione thiol-disulfide status are not involved in the control mechanisms which operate during fertilization or the first cell division cycle in the sea urchin egg.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Propranolol induces polyspermy during sea urchin fertilization

Propranolol, a β-adrenergic receptor blocker, is found to induce polyspermy in sea urchin eggs. Unfertilized sea urchin eggs treated for 10 min with 50 μM of propranolol, and then inseminated, become polyspermic and show a fertilization envelope which is barely visible to the light microscope. Examination of treated eggs by transmission and scanning electron microscopy shows that the drug does not alter the cortex of the unfertilized egg. However, after insemination an incomplete cortical reaction occurs. This might well account for both polyspermy and the defective elevation of the fertilization envelope. Since the effects of the drug are reversed by simultaneous treatment with adrenalin, perhaps propranolol interferes with the monoaminergic system that has been proposed to be active. The involvement of the monoaminergic system in the fertilization process is present in the sea urchin egg. © 1996 Wiley-Liss, Inc.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

Acid release,cytoplasmic alkalinization,and chromosome condensation during sea urchin fertilization and amine-lnduced parthenogenesis

We have studied the relationship between acid release, cytoplasmic alkalinization, and the extent of chromosome condensation during parthenogenetic activation of sea urchin eggs. The relative rate of acid release in Strongylocentrotus purpuratus eggs was determined from pH measurements of egg suspensions. Acid release in inseminated eggs began after a lag of 0.4 min and the relative rate increased 108-fold, declined, and release was essentially complete by 8-min postinsemination. An average of 3.8 ± 0.23 × 10^?12moles H⁺ cell^? was released as determined by backtitration with NaOH. Acid release characteristics of eggs parthenogenetically activated with either NH₄C1, methylamine ethylamine, n-propylamine, n-butylamine, or benzylamine were qualitatively similar. There was no detectable lag peroid and the increase in relative rate of acid release was directly proportional to the carbon number of the amine used, eg, from 8.3-fold methylamine to 470-fold with benzylamine. The total equivalents of acid released ranged from 0.50–8.2 × 10^?12 moles H⁺·cell^? in direct proportion to the concentration of amine used. The degree fo cytoplasmic alkalinization induced as a function of methylamine and benzylamine concentration was determined by pH measurements fo egg homogenates; egg cultures were also prepared for microscopic examination of chromosome condensation. None of the eggs had condensed chromosomes at 0.5-mM methylamine whereas a cytoplasmic alkalinization of 0.6 pH units was observed. Increased methylamine levels up to 10mM resulted in chromiosome condensation in only 20% of the eggs. A similar result was found with benzylamine. We conclude that acid release and cytoplasmic alkalinization during chemical parthenogenesis are insufficient to mimic sperm induction of chromiosome condensation and suggest that an additional factor(s) is required for chromosome condensation by low concentration of amines.     

Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs

Investigations were conducted in an effort to determine the origin of the membrane comprising the male pronuclear envelope of inseminated sea urchin eggs. The events of fertilization in zygotes treated with 200 μg/ml of puromycin are not impaired even though incorporation of [³H]leucine is inhibited up to 80% when compared to control specimens. Developing male pronuclei in zygotes treated with puromycin form nuclear envelopes structurally similar to and within the same period as controls. In puromycin-treated and untreated zygotes morphologically recognizable portions of the sperm nuclear envelope are incorporated into the structure of the male pronuclear envelope. Pronuclear development was also examined in inseminated ova where most of the endoplasmic reticulum (ER) was confined to a specific area of the zygote. Eggs were centrifuged in order to stratify their organelles into specific layers (stratified eggs); with further centrifugation stratified eggs are bisected to form nucleate (rich in ER) and nonnucleate halves (containing little ER). Observations of inseminated stratified eggs and nucleate and nonnucleate halves demonstrate an inverse relation between the amount of ER present in the vicinity of a reorganizing sperm nucleus and the time it takes to form the male pronuclear envelope. Computation of the maximum quantity of membrane in the male pronucleus that may be derived from the sperm nuclear envelope is approximately 15%. These investigations suggest that a major portion of the male pronuclear envelope is derived from endoplasmic reticulum within the egg and only a small portion (up to 15%) originates from the sperm nuclear envelope.     

Studies on the interactions of sperm with the surface of the sea urchin egg

We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbacia punctulata eggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. purpuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence of S. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. purpuratus sperm bind to an A. punctulata egg in the presence of S. purpuratus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. punctulata eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulata sperm to fuse and fertilize with A. punctulata eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpuratus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. purpuratus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process.     

Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strongylocentrotus purpuratus embryos

The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca²⁺- and Mg²⁺-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Voltage clamp studies of fertilization in sea urchin eggs: I. Effect of clamped membrane potential on sperm entry,activation, and development

To analyze the role of the activation potential (a positive shift of the membrane potential which occurs following sperm attachment) in fertilization and development of the sea urchin egg, unfertilized Lytechinus variegatus eggs were voltage clamped at membrane potentials (E_m) from +20 to ?90 mV, and then inseminated. Either a fast two electrode voltage clamp, or a single electrode switched voltage clamp was used. The clamp was maintained for 3 to 15 min after initiation of a conductance increase. At E_m more positive than +18 mV, even though many sperm may attach, the egg remains completely inert (Jaffe, Nature (London)261, 68–71, 1976). At E_m from +17 to ?90 mV, all inseminated eggs elevate normal fertilization envelopes, although substantially increased concentrations of sperm are required at E_m from +17 to +12 mV. Whether cleavage occurs depends on the clamped E_m. When clamped at E_m from +17 to ?25 mV, 100% of activated eggs cleave. However, when clamped at E_m from ?26 to ?75 mV the percentage of activated eggs which cleave progressively decreases. At clamped E_m between ?76 and ?90 mV, none of the activated eggs cleave. All monospermic voltage clamped eggs that cleave develop to normal swimming blastulae. In all eggs that fail to cleave (clamped at E_m more negative than ?30 mV), sperm penetration is blocked, the sperm is lifted off the egg surface as the fertilization envelope rises, and a sperm aster never forms. Preventing formation of the fertilization envelope by prior disruption of the vitelline layer with dithiothreitol does not promote entry of the sperm. In conclusion, preventing the depolarization normally associated with fertilization suppresses sperm entry in the sea urchin egg, yet activation proceeds. Present evidence suggests an effect of the electrical field across the plasma membrane in suppressing sperm entry.     

Phospholipid metabolism following fertilization in sea urchin eggs and embryos.

Phospholipid metabolism during early development was examined in the sea urchins Stronglyocentrotus purpuratus and Lytechinus pictus. Transport of ³H-choline was stimulated fivefold following fertilization in both species. However, the actual percent incorporation of labeled precursors into phospholipids from the TCA soluble pool did not change at fertilization. There was a slight increase in transport of ¹⁴C-ethanolamine at fertilization but again there was no change in its percent incorporation into phospholipids. When eggs were preloaded with ³H-choline or ¹⁴C-ethanolamine and fertilized, the eggs or embryos showed similar patterns of incorporation into phospholipids. There was no significant change in the percent phosphorylation of choline in fertilized or unfertilized eggs.An investigation was made of the activity of choline kinase, the first enzyme in the biosynthesis of phosphatidylcholine. This enzyme was found to have similar activities in fertilized and unfertilized eggs using a variety of homogenization media. The activity of choline kinase was found to decrease slightly in activity at fertilization and reach a maximum activity by gastrula.These results indicate that there is no activation of phospholipid synthesis at fertilization of sea urchin eggs. Apparent increased incorporation actually reflects increased transport of precursors and not de novo synthesis.     

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.     

Translational diffusion in the plasma membrane of sea urchin eggs

Translational diffusion in the plasma membrane of individual egg cells from the sea urchin species Paracentrotus lividus has been studied by fluorescence microphotolysis (FM). In order to probe the lipid phase of the membrane, procedures have been worked out by which the fluorescent analog 3,3′-dioctadecyl-oxatricarbocyanine (C₁₈diO) can be incorporated into the membrane. In the unfertilized egg a fraction R = 0.9 of C₁₈diO was mobile having an apparent diffusion coefficient of D = 6.0 × 10^?9 cm² sec^?1. Fifteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 2.7 × 10^?9 cm² sec^?1, respectively. In order to study diffusion of membrane proteins, procedures have been worked out by which the cell surface can be labeled with fluorescein-isothiocyanate (FITC). FITC binds to both the plasma membrane and the vitelline layer. Together with the vitelline layer two-thirds of the FITC-fluorescence could be removed from the egg surface. Gel electropherograms of isolated egg cortices showed various protein bands; however, only two of the protein bands were labeled with FITC. In the unfertilized egg a fraction R = 0.9 of the FITC-labeled membrane proteins was mobile having an apparent diffusion coefficient of D = 35 × 10^?11 cm² sem^?1. Fiteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 7.0 × 10^?11 cm² sec^?1, respectively. FITC-labeled proteins of the fertilization envelope were immobile. Our studies have shown (i) that the egg surface can be fluorescently labeled without blocking fertilization and early development, (ii) that the plasma membrane of unfertilized eggs is a fluid environment permitting a rapid movement of lipids and proteins, and (iii) that after fertilization a substantial degree of lipid and protein mobility is maintained.     

Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs

The membrane potentials of sea urchin (Hemicentrotus pulcherrimus) eggs before and after fertilization and their changes during the membrane elevation induced by intracellular electrical stimulation were recorded in solutions of various ionic compositions. Upon fertilization, the membrane potential (?10 mV) depolarized and reversed polarity by a few mV, then gradually returned to a new steady level ranging between ?50 and ?60 mV. The activation potential is closely associated with a transient increase in the membrane permeability. The potential of the unfertilized egg is hyperpolarized by monovalent anions (Br^?, Cl^? and NO₃^?) and depolarized slightly by K⁺. In contrast, the membrane of the fertilized egg is markedly depolarized by K⁺. Suppression of depolarization associated with an increase of the membrane permeability was recorded in Na-free medium (Tris-HCl). The selective increase in permeability to monovalent anions is thought to alternate with the selective increase in permeability to K⁺through the mediation of a transient increase of Na⁺-permeability at the time of fertilization. No causal relationship between the membrane elevation and the depolarization was established because the breakdown of the cortical granules occurs without depolarization or an increase in membrane permeability.     

Phosphate transport in fertilized sea urchin eggs. I. Kinetic aspects

Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10^?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding K_i values ranging from 10.5 to 14.1 × 10^?6 M in comparison to the corresponding apparent K_M (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10^?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10^?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO₄^??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO₄^?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10^?4 M), and adenosine triphosphate (1–5 × 10^?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca⁺⁺ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca⁺⁺ and Mg⁺⁺ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′₂ = 6.14 and for pK′₃ = 10.99, at 15°C and phosphate at infinite dilution.