Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.     

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.     

High pH-induced acrosome reaction and Ca2+ uptake in sea urchin sperm suspended in Na+-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca2+ and Na+ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na+ in this reaction. Sperm were suspended in Na+-free seawater and the percentage of acrosome reaction and the amount of Ca2+ uptake were determined as a function of external pH. High pH (9.0) in Na+-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca2+ uptake. Both the percentage of acrosome reactions and the amount of Ca2+ uptake were similar to those induced by either jelly or pH 9 in Na+-containing seawater. On the other hand, the absence of Na+ in seawater inhibits jelly from inducing Ca2+ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na+ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca2+ was strictly required since sperm did not react in Ca2+-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca2+ uptake in complete and Na+-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca2+ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca2+ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca2+ uptake induced by jelly or pH 9 in complete seawater.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Protein tyrosine kinase activity of eggs of the sea urchin Strongylocentrotus purpuratus: the regulation of its increase after fertilization

Protein tyrosine kinase activity in eggs of the sea urchin, Strongylocentrotus purpuratus, increased two- to fourfold as early as several min after fertilization at 8-10 degrees C. Artificial activation of eggs with the divalent cation ionophore, A23187, or with butyric acid induced the increase in enzyme activity. The transfer of eggs to seawater containing either no Na+ or 50 mM Na+ and 10(-4) M amiloride immediately after fertilization did not block the increases in enzyme activity. When eggs were activated with seawater containing NH4OH, enzyme activity did not increase at 1 hr after activation, although the increased activity was detected at 3 hr after activation. Increased enzyme activity also was observed in enucleated egg fragments activated with butyric acid. Puromycin and emetine, inhibitors of protein synthesis, also did not inhibit the initial increases of enzyme activity after fertilization. These results demonstrated that the increased protein tyrosine kinase activity observed after fertilization of S. purpuratus eggs can be initiated independent of various other known events such as fusion with sperm cells and protein and DNA synthesis.     

Effect of Na+-free seawater on energy metabolism in sea urchin spermatozoa with special reference to coenzyme A and carnitine derivatives

When the dry sperm of sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial seawater, they became motile and the level of ATP decreased. However, after dilution in Na⁺-free seawater, the sperm were still immotile and the ATP level remained constant. CoA, carnitine, and their derivatives as intermediates of lipid metabolism were also measured in sperm. The levels of CoA derivatives were much higher than those of carnitine derivatives. The free and acid-soluble CoA level decreased and the long-chain acyl-CoA level increased after dilution in artificial seawater, However, they did not change after dilution in Na⁺-free seawater. The levels of carnitine derivatives hardly changed after the dilution in both sorts of artificial seawater, The respiratory rate was extremely low in Na⁺-free seawater, Furthermore, concentrations of pyruvate, lactate, citrate, and malate remained essentially constant after dilution in the Na⁺-free seawater, whereas they changed markedly after dilution in the artificial seawater.     

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.     

The Role of External Potassium Ion in Activation of Sea Urchin Spermatozoa

When sperm of the sea urchin, Hemicentrotus pulcherrimus, are diluted into K⁺-free seawater, the pH of the suspension gradually decreases, whereas a rapid decline in pH is observed following dilution into regular seawater. Sperm motility and respiration are also activated after dilution into K⁺-free seawater, but levels of activity are less than those observed following dilution into regular seawater. Upon addition of 10 mM K⁺ to K⁺-free seawater, rapid acid release occurs and motility and respiratory rate in sperm are reactivated. The effect of K⁺ on respiration was competitive with respect to the external Na⁺ concentrations. Harmaline, a potent inhibitor of Na⁺/K⁺-ATPase, causes a decrease in movement and respiration of the sperm. Harmaline does not inhibit the rapid decline in pH, although it depresses the release of acid from mitochondria. These results suggest that external K⁺ plays an important role in intracellular alkalinization of sea urchin sperm.     

Alteration of intracellular [Ca2+] in sea urchin sperm by the egg peptide speract. Evidence that increased intracellular Ca2+ is coupled to Na+ entry and increased intracellular pH

The egg peptide speract increases intracellular pH (pHi) and cyclic nucleotides in sperm of the sea urchin Strongylocentrotus purpuratus by a mechanism dependent on seawater Na+ but not Ca2+ (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 2235-2241; Repaske, D. R., and Garbers, D. L. (1983) J. Biol. Chem. 258, 6025-6029). Using the Ca2+ indicators quin2 and indo-1, we show that speract stimulates a transient rise in intracellular [Ca2+] ([a2+]i) when millimolar Ca2+ is present in seawater. The rise is increased and extended by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), which also enhances 22Na+ uptake with or without Ca2+. Without MIX, speract initiates a rise in [Ca2+]i that peaks within approximately 5 s and decreases with a t1/2 of approximately 9 s. Activation of Na+:H+ exchange without speract by either Na+ addition to sperm in Na+-free seawater (NaFASW) or by monensin also increases [Ca2+]i, but neither change is transient. Inhibition of Na+:H+ exchange by increased seawater [K+] prevents the rise in [Ca2+]i initiated by either speract or Na+ addition to sperm in NaFASW. Increasing pHi by adding 10 mM NH4+ or by addition of Li+ to sperm in NaFASW does not increase [Ca2+]i. The data suggest that speract binding leads to rapid activation of Na+:H+ exchange; and, as a consequence, [Ca2+] entry increases transiently through either Na+:Ca2+ exchange or else through a verapamil-insensitive Ca2+ channel. MIX prevents the inactivation of this entry mechanism.     

A study of factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata.

Several factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata, have been investigated quantitatively using a simple substrate film technique to monitor extension of the acrosomal process by electron microscopy. Verification of typical acrosomal process formation has been accomplished using thin sections. Sperm were found to undergo the acrosomal reaction in artificial sea water in the absence of egg jelly coat at pH values above 9.6. In the presence of egg jelly a high percentage of sperm react at pH 8.6. At this pH, the fraction of sperm that undergo the acrosomal reaction is directly proportional to the concentration of egg jelly. The Ca²⁺ ionophore A23187 induces the acrosomal reaction in the absence of egg jelly at pH 8.6. The proportion of sperm that react is dependent on the concentration of ionophore and on the concentration of Ca²⁺ in the medium. Pretreatment of sperm with low levels of La³⁺ ion, which is known to be a Ca²⁺ ion antagonist, results in inhibition of egg jelly induction of the acrosomal reaction. These findings suggest that there are marked similarities between the acrosomal reaction in sea urchin sperm and membrane fusion dependent secretory processes in other cell types.     

Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract

Sea urchin sperm motility can be activated by alkalinization of the internal pH, and previous studies have shown that the internal pH can be regulated by a voltage-sensitive Na+/H+ exchanger present in the flagellar plasma membrane. In this study, the effects of speract, a peptide purified from egg conditioned media, on the Na+/H+ exchange were investigated. Evidence presented indicates that speract activates K+ channels in the flagellar membrane and modulates the Na+/H+ exchange activity through resultant changes in membrane potential. In the presence of tetraphenylphosphonium, a lipophilic ion, or high external Na+, the isolated flagella were depolarized, and Na+/H+ exchanger was inhibited. Speract and valinomycin, a K+ ionophore, were able to reactivate 22Na+ uptake, H+ efflux, and alkalinization of intraflagellar pH under either of the depolarizing conditions. Membrane potential measurements using 3,3'-dipropylthiodicarbocyanide iodide indicated repolarization by either speract or valinomycin. The speract-induced voltage changes did not require Na+ but were sensitive to [K+]. Thus, speract induced a slight depolarization in Na+-free seawater with 10 mM K+ but a hyperpolarization with 2 mM K+. Further support for the activation of K+ channels in the flagella was the 2-5-fold stimulation of K+ efflux induced by speract as measured with a K+ electrode. The ionic selectivity of the speract-activated channel assessed by voltage measurements was K+ greater than Rb+ greater than Cs+. The half-maximally effective concentration of speract was about 0.2 nM. That the H+ and K+ efflux in response to peptide was receptor-mediated was confirmed by the use of speract or resact on intact sea urchin spermatozoa, where the peptides were found to stimulate K+ efflux and to reverse the tetraphenylphosphonium inhibition on H+ efflux only in the homologous spermatozoa. Modulation of the voltage-sensitive Na+/H+ exchange by egg peptides, therefore, appears to be indirect and is coupled through its action on membrane potential.     

The dissociation of exocytosis and respiratory stimulation in leucocytes by ionophores.

By exploiting the unique characteristics of three ionophores, experimental conditions were found which permit the dissociation of respiratory stimulation from secretion in polymorphonuclear leucocytes. A marked stimulation of respiration was produced by ionophore X537A, which binds and transports both alkali-earth and alkali cations. The stimulatory activity of this ionophore was the same at either high or low Na+/K+ ratios in the medium and was virtually unaffected by extracellular Ca2+. A slight stimulation of oxygen consumption was also caused by the K+-selective ionophore valinomycin and by ionophore A23187, which complexes and transfers bivalent cations. Ionophore X537A and valinomycin were unable to stimulate selective release of granuleassociated beta-glucuronidase and gradually increased cell fragility, as monitored by increased leakage of lactate dehydrogenase. Ionophore A23187 slightly increased exocytosis of beta-glucuronidase. In a Mg2+-free medium, Ca2+, added simultaneously with ionophore A23187, greatly enhanced respiration and secretion of the granule enzyme. If Ca2+ was added a few minutes after the ionophore, exocytosis occurred, but no respiratory burst was observed. If the latter experiment was repeated in the presence of extracellular Mg2+, both secretion and respiration were stimulated. This effect was not produced by Mn2+ or Ba2+. It is proposed that Ca2+ is required for triggering selective secretion of granule enzymes from leucocytes is caused by an intracellular redistribution of cations, which may invovle Mg2+-dependent mechanisms.     

The role of Ca2+ and protein kinase C in the acrosome reaction of giant panda (Ailuropoda melanoleuca) spermatozoa

Fresh semen was collected from adult male giant pandas and the role of Ca2+, Ca2+ ionophore A23187 and protein kinase C (PKC) in sperm motility and acrosome reaction (AR) was assessed by lens culinaris agglutinin conjugated with fluorescein isothiocyanate (FITC-LCA) labeling and transmission electron microscopy. The AR in giant panda spermatozoa was characterized by vesiculation of the outer acrosomal membrane through its invagination. Both the sperm motility and the AR rate decreased significantly (p < 0.05) in Ca2+-free and low Ca2+ medium. The addition of 10 microM Ca2+ ionophore A23187 potently stimulated AR. After incubation for capacitation, the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated AR in a dose-dependent manner and its effect could be overcome by the PKC inhibitor staurosporine. These results suggest that Ca2+ and PKC play an important role in the sperm acrosome reaction of the giant panda.     

Purification of sea urchin sperm bindin by DEAE-cellulose chromatography

A procedure for purifying bindin from sperm of the sea urchin Strongylocentrotus purpuratus is presented in detail. The impure bindin, dissolved in 4 M urea, 50 mM sodium phosphate, pH 6.6, is adsorbed to DEAE-cellulose and eluted wit 4 M urea, 650 mM sodium phosphate, pH 6.6. The purified bindin is not contaminated with tubulin or histone HI. A precipitate of this DEAE-purified bindin, made by dialysis into Ca2+-free seawater and natural seawater, is a species-specific agglutinin of unfertilized eggs. This method of obtaining consistently pure preparations of bindin will aid in the analysis of its role in fertilization.     

The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.     

Regulation of internal pH of sea urchin sperm. A role for the Na/K pump

In the absence of sodium, sea urchin sperm have an acidic internal pH. The addition of sodium, lithium, or ammonium, but not of potassium ions, induces an internal alkalization. If potassium is added in the presence of sodium, a further alkalization is obtained; in contrast, potassium addition in presence of Li+ or NH+4 does not change the internal pH. The K+-induced pHi change is inhibited by ouabain and when sperm are depleted of their ATP. A large part of the potassium influx is stimulated by Na+, but not Li+, and inhibited by ouabain and cellular ATP depletion. We conclude that activity of Na/K-ATPase pumps located in the plasma membrane of sea urchin sperm could play a role in regulating the internal pH of sea urchin sperm by recycling sodium ions that enter the cell through Na/H countermovements.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Involvement of zinc in the regulation of pHi, motility, and acrosome reactions in sea urchin sperm

When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions.     

Activation of amino acid uptake at fertilization in the sea urchin egg. Requirement for proton compartmentalization during cytosolic alkalosis

The comparative importance of the release of intracellular ionic calcium, Na+/H+ exchange and cytosolic alkalosis as activator signals was studied on the development of amino acid uptake at fertilization in sea urchin eggs. We show that, once stimulated, the rate of valine uptake is greatly dependent upon intracellular pH. Suppression of the Na+/H+ exchange at the time of activation, by applying ionophore (A23187) in sodium-free artificial sea water (ONaASW), inhibits the development of valine influx. This cannot be restored by a further (30 min later) alkalosis by transferring eggs into sea water. Suppressing the alkalosis in the presence of Na+/H+ exchange at fertilization by simultaneous addition of acid into sea water results in activation of the amino acid carrier which exhibits an increased rate of transport as soon as the eggs are replaced in sea water at pH 8.0. The absence of alkalosis in eggs activated in ONaASW can be counterbalanced either by adding NH4Cl 10 mM or by transfer into ASW at pH 9.0 at activation. Ammonia-treated eggs absorbed amino acid as controls, whereas eggs in sea water at pH 9.0 failed to develop a valine uptake system, suggesting that ammonia can completely replace the effect of Na+/H+ exchange. Furthermore, addition of NH4Cl immediately before fertilization conceals the Na+/H+ exchange but stimulates valine uptake as in controls. These data suggest that: the occurrence of the intracellular calcium increase alone is not sufficient for the develpment of the amino acid transport system; cell alkalinization at fertilization derives from the cytoplasmic membrane-located Na+/H+ exchange and an inward movement of protons into a cortical acidic compartment, which is discussed.     

Oral—aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral–aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral–aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oralaboral axis during sea urchin development.