Bennett M. Shapiro

The acrosome reaction of Strongylocentrotus purpuratus sperm. Ion requirements and movements.

Abstract The acrosome reaction of sperm of the sea urchin, Strongylocentrotus purpuratus, is accompanied by ion movements. When the reaction is induced by the addition of egg jelly to sperm suspended in sea water, there is an acid release and an uptake (or exchange) of calcium ions. Verapamil and D600, drugs which block Ca2+ channels, inhibit induction of the acrosome reaction, acid release, and 45Ca2+ uptake; this inhibition is reduced at higher concentrations of external Ca2+. Although acid release correlates temporally with extension of the acrosome filament, 45Ca2+ uptake continues after the acrosome reaction has been completed. Neither the acrosome reaction nor acid release is inhibited by cyanide, azide, dinitrophenol (DNP), or carbonyl cyanide m-chlorophenylhydrazone (CCCP), whereas these metabolic inhibitors partially inhibit Ca2+ uptake. Tetraethylammonium (TEA) chloride, an inhibitor of delayed axonal potassium currents, inhibits the acrosome reaction. An increase in 86Rb+ permeability accompanies the acrosome reaction, suggesting that movement of K+ is an important effector of the reaction. In support of this, the acrosome reaction may be triggered with nigericin, an ionophore that catalyzes the electrically neutral exchange of K+ and H+ across membranes. Induction of the acrosome reaction with nigericin can occur with either Na+ or K+ as the predominant external monovalent cation, while with jelly it requires external Na+. With nigericin, there is a delay in acid release, Ca2+ uptake, and filament extension, all of which follow a transient proton uptake. Taken together, these data suggest that triggering of the acrosome reaction involves linked permeability changes for monovalent and divalent ions.

Metabolite channeling: A phosphorylcreatine shuttle to mediate high energy phosphate transport between sperm mitochondrion and tail

Energy utilization by the flagellum of motile sea urchin sperm is tightly coupled to the rate of energy production by the mitochondrion. This tight coupling depends upon the transport of high energy phosphate (P) from mitochondrion to axoneme, which we propose to be mediated by a phosphorylcreatine shuttle. The shuttle employs distinct mitochondrial and axonemal creatine kinase isozymes, the latter being a novel creatine kinase of 145 kd. To examine whether P is directed to the tail by such a shuttle, we inactivated creatine kinase specifically with fluorodinitrobenzene. Creatine kinase inactivation led to an inhibition of coupled, but not uncoupled, respiration and affected the pattern of sperm motility as predicted for the disruption of an obligatory link in P transport.

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates 45Ca2+ and 22Na+ uptake, and K+ and H+ efflux. H+ efflux and 22Na+ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K+ loss, as measured either by 42K+ efflux or K+-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K+ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K+ channels, inhibits the acrosome reaction half-maximally, K+ movements or alterations of K+-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the 45Ca2+ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN−, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca2+. The extracellular Na+ concentration affects Ca2+ fluxes: sperm placed into 5 mM Na+ seawater have enhanced 45Ca2+ uptake, but do not undergo the acrosome reaction, unless 30 mM Na+ is also added. Low Na+ concentrations lead to spontaneous triggering, by allowing for both Ca2+ influx and Na+-dependent H+ efflux. At least one early Ca2+ requirement precedes the Na+ and H+ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca2+, then washed and incubated with jelly in the presence of Ca2+, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na+ seawater, low pH, or elevated K+), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca2+ is required, but that precedes the Na+ and H+ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.

When sperm meets egg: biochemical mechanisms of gamete interaction.

Publisher Summary This chapter discusses the biochemical mechanisms of gamete interaction. The chapter focuses on three principal subjects: (1) the effect of the egg on the sperm; (2) the contact between sperm and egg; and, (3) the effect of the sperm on the egg. The most dramatic morphological response of the sperm to the egg is the acrosome reaction, in which a change that is a prerequisite for fertilization occurs in the head of sperm from many species. In mammals, sperms undergo a process known as capacitation before they respond to the egg. The moment of union of sperm and egg is the most dramatic of the events surrounding fertilization. The locus on the egg for interaction with sperm varies from species to species. Several enzymes have been implicated in the process of sperm–egg interaction. These enzymes play a role in sperm–egg membrane fusion, for it could lead to a transient state of membrane instability. The most dramatic effect of fertilization on the egg surface is the induction of the cortical reaction. The properties of the egg plasma membrane change enormously after cortical granule exocytosis.

Sequential biochemical and morphological events during assembly of the fertilization membrane of the sea urchin

The fertilization membrane of Strongylocentrotus purpuratus undergoes changes in morphology, solubility, and permeability during the process of hardening. As the fertilization membrane elevates from the egg surface, it retains casts of the tips of the microvillous processes of the plasma membrane. The dome-shaped microvillar casts become angular at the same time that the fertilization membrane becomes resistant to solubilization in mercaptan solutions. 2-4 min after this morphological and chemical transition, the fertilization membrane becomes impermeable to the lectin conconavalin A, as monitored by binding of 125I- or fluorescein-labeled concanavalin A. Glycine ethyl ester inhibits the changes in morphology, solubility, and permeability, whereas sodium sulfite inhibits only the permeability block and resistance to solubilization by mercaptans. Parthenogenetic activation with the divalent ionophore, A23187, elicits fertilization membrane elevation more rapidly than does activation by fertilization; however, the morphological and permeability changes characteristic of hardening proceed more slowly. Elevation and hardening of the fertilization membrane thus appear to be discrete, multiple-step assembly processes that occur in fixed sequence, with kinetics that are affected by the mechanism of cortical granule exocytosis.

Ovothiol replaces glutathione peroxidase as a hydrogen peroxide scavenger in sea urchin eggs

Despite its potential toxicity, H2O2 is used as an extracellular oxidant by Stronglylocentrotus purpuratus eggs to cross-link their fertilization envelopes. These eggs contain 5 mM 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine (ovothiol C), which reacts with H2O2. In consuming H2O2 and being reduced by glutathione, ovothiol acts as a glutathione peroxidase and replaces the function of the enzyme in eggs. The ovothiol system is more effective than egg catalase in destroying H2O2 at concentrations produced during fertilization and constitutes a principal mechanism for preventing oxidative damage at fertilization.

After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.

Interactions between sperm and sea urchin egg jelly

The addition of egg jelly to sea urchin sperm induces multiple changes in morphology and behavior. When jelly is added to sperm diluted in seawater, the acrosome reaction is triggered, the mitochondrion rounds up, the internal pH is transiently alkalinized and then reacidified, and respiration becomes uncoupled and rapidly decreases. Sperm also become unable to fertilize eggs within a few minutes after jelly addition. In order to explore in more detail the effect of egg jelly on sperm, we have studied the response to jelly in the presence of inhibitors of the acrosome reaction. When jelly is added to sperm under conditions which are inhibitory for the acrosome reaction, an alkalinization takes place without the subsequent reacidification, the mitochondria remain coupled, and respiration and intracellular ATP levels remain high. Sperm viability is prolonged by some of these conditions, but not others. The addition of jelly to sperm in the absence of calcium elicits an internal alkalinization but no other rapid change in sperm physiology. The capacity of egg jelly to alter sperm physiology even when the overall acrosome reaction is inhibited indicates that some of the physiological changes either are early events in the triggering pathway that happen before the inhibitory step or are unrelated to the acrosomal reaction itself. The reacidification of the internal pH, the uncoupling and decrease of the respiration, and the decrease of the ATP levels might be linked together by the large influx of calcium that occurs after the acrosome reaction.

Limited proteolysis of some egg surface components is an early event following fertilization of the sea urchin, Strongylocentrotus purpuratus☆

The surface proteins of eggs from Stronglocentrotus purpuratus were labeled with 125I by lactoperoxidase-catalyzed iodination. The eggs were examined after solubilization and disaggregation in sodium dodecyl sulphate (SDS) by electrophoresis on SDS-polyacrylamide slab-gels. Seventy-five percent of the label was found in material with a molecular weight greater than 130,000. About 5% of the radioactivity was excluded from the gels. Upon fertilization, embryos show a redistribution of the radioactively labeled species. There is a decrease in the amount of very high molecular weight material but an increase (35–40%) in material excluded from the gel. In addition, new radioactive bands of lower molecular weight are found. This change of distribution in the radioactive bands is blocked by inclusion of soybean trypsin inhibitor either before or immediately after fertilization, which completely inhibits the cortical granule protease. The disappearance of high molecular weight components is prevented by treatment of the eggs with procaine during fertilization, although the appearance of low molecular weight bands (approximately 20,000 and 30,000) is not completely blocked by procaine treatment. Parthenogenic activation of eggs by butyric acid or partial metabolic activation by ammonia each leads to changes in the egg surface proteins which are similar but not identical to those seen after fertilization. The data suggest that the labeling occurs on the vitelline membrane, plasma membrane and jelly layer. The possible significance of limited proteolysis in fertilization is discussed.

31P-NMR analysis of sea urchin sperm activation: Reversible formation of high energy phosphate compounds by changes in intracellular pH

31P-NMR has been used to estimate the internal pH (pHi) of sperm from the sea urchin Strongylocentrotus purpuratus. The values for pHi obtained from the chemical shift of inorganic phosphate agree well with those obtained from amine accumulation. At low pHi, when sperm are quiescent (immotile and non-respiring), they accumulate phosphocreatine (PCr), but have a low level of inorganic phosphate (Pi). Conversely, when the pHi is elevated, sperm respiration and motility are activated, PCr is decreased and Pi is increased. This change is reversible upon decrease of the pHi, whereupon respiration and motility are arrested, Pi disappears and PCr increases. We conclude that the overall balance of energy metabolism, and thus the phosphate potential, of sea urchin sperm are under the control of the pHi.