A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm.

The acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca(2+) ([Ca(2+)](i)). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca(2+)](i) elevation lasts minutes and involves two Ca(2+) permeable channels. Both the opening of the second channel and the onset of the AR occur approximately 5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca(2+) from sea water or addition of Ca(2+) channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a "rapid" (t(1/2) = 3--15 s) decrease in [Ca(2+)](i) and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH(4)Cl and either EGTA, Co(2+), or Ni(2+) 5 s after egg factor prevents the partial inhibition of the evoked pH(i) change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca(2+)](i) caused by the second Ca(2+) channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca(2+) uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca(2+) channel.

The acrosome reaction in digitonin-permeabilized sea urchin sperm in the absence of the natural inducer.

In many species, the acrosome reaction of sperm is an obligatory step in fertilization. Increases in [Ca2+]i and pHi, activation of adenylyl cyclase and inositol trisphosphate generation accompany the egg jelly-induced acrosome reaction of sea urchin sperm. The signaling mechanisms involved are unknown. We used digitonin, a cholesterol-complexing compound, to selectively permeabilize the plasma membrane of sea urchin sperm suspended in a medium that mimics the cytosolic ion composition. Within 6 to 8 min, 30 to 50 microM digitonin allowed incorporation of the membrane-impermeant dye Hoechst 33258 into the sperm, staining exclusively the nucleus. No alterations in sperm morphology were caused by digitonin at the concentrations used, however, it irreversibly permeabilized the plasma membrane. Permeabilized sperm retained lactate dehydrogenase and actin. When incubated in Ca(2+)-containing permeabilization buffer (pH 7.8), sperm were capable of undergoing spontaneously the acrosome reaction; this reaction was pH dependent and displayed an absolute Ca2+ requirement. Electron microscopy indicates that the acrosome reaction undergone by permeabilized sperm resembled that induced by egg jelly. Additionally, rhodaminyl-phalloidin staining of sperm reacted under permeabilizing conditions revealed a fluorescent filament in the acrosomal tubule region, demonstrating the occurrence of actin polymerization. Thus, in permeabilized sperm the machinery necessary to perform a [Ca2+]i- and pHi-sensitive acrosome reaction is functionally preserved. Permeabilized sperm offer new avenues to study the molecular bases of the sea urchin sperm acrosome reaction.

Ionic bases of the membrane potential and intracellular pH changes induced by speract in swollen sea urchin sperm.

Signal transduction initiated by the egg peptide, speract, in sea urchin sperm is not fully understood. Hypotonically swollen sperm are a suitable model to study peptide signal transduction. Ion substitution experiments now indicate (i) that the permeability to Na+, Ca2+, and Mg2+ contributes to the sperm resting membrane potential; (ii) the repolarization induced by nM concentrations of speract is Na+ dependent and mediated by an as yet unidentified channel; (iii) the depolarization triggered by nM concentrations of speract involves Ca2+ channels since it is Ca(2+)-dependent and blocked by Co2+ and Ni2+, two Ca2+ channel blockers; (iv) hyperpolarizing swollen sperm with valinomycin increases intracellular pH (pHi) in the same way as speract, thus the speract-induced hyperpolarization may be responsible for the pHi increase.

A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm.

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), a K(+)-dependent transient hyperpolarization which may involve K+ channels, and a depolarization which depends on external Ca2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K(+)-free seawater raised the pHi, caused a small increase in 45Ca2+ uptake, and triggered some AR. When the cells were depolarized with KCl (30 mM) 40-60 sec after the induced hyperpolarization, the pHi decreased and there was a significant increase in 45Ca2+ uptake, [Ca2+]i, and the AR. This waiting time was necessary in order to allow the pHi change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pHi, may regulate Ca2+ transport systems involved in this process. Because of the key role played by K+ in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake.

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.

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.

Response of isolated sperm plasma membranes from sea urchin to egg jelly.

The acrosome reaction in sea urchin sperm is induced by a glycoprotein jelly surrounding the egg and is accompanied by changes in ion permeability of sperm plasma membrane. In an attempt to learn what membrane components are involved in the response to jelly, we have begun to reassemble sperm membrane components into artificial membranes and assay for permeability changes mimicking those that occur in sperm. Jelly in sea water at concentrations that induce the acrosome reaction did not significantly change 45Ca2+ uptake of sonicated unilamellar vesicles made with soybean lipid only (ratio jelly:control uptake = 1.08 +/- 0.36 SD, n = 21). Experiments with pure lipid planar bilayers made with soybean lipid or a lipid extract from sperm and held at various voltages, also did not reveal substantial permeability changes at comparable jelly concentrations. Thus, jelly by itself does not change the conductance of a pure lipid bilayer. In contrast, significant (P----0.0005, t test for two sample means) 45Ca2+ uptake was observed with vesicles made by cosonicating soybean phospholipids and Strongylocentrotus purpuratus sperm membranes isolated by the method of Cross, N. L. [1983, J. Cell Sci. 59, 13-25] (ratio jelly: control uptake = 1.51 +/- 0.75, n = 20, 16 positive out of 20 experiments). The calcium uptake response of the mixed vesicles was also species-specific: it did not occur with jelly from Arbacia punctulata (ratio Arbacia jelly: control = 1.18 +/- 0.51; ratio Strongylocentrotus jelly: control = 1.71 +/- 0.97, n = 10; P----0.025, paired t statistic). Vesicles made with soybean lipid and an octyl glucoside extract of sperm membranes also responded to jelly with increased 45Ca2+ uptake. Our results indicate that we have the starting conditions to isolate and characterize the sperm membrane components that participate in the egg jelly induced permeability changes.