Robert W. Schackmann

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.

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.

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.

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.

Ionic regulation of sea urchin sperm motility, metabolism and fertilizing capacity.

In order to pursue the significance of the ionic regulation of sea urchin sperm behaviour, alterations in the cation composition of sea water were tested for their effects on sperm fertilizing capacity. Nearly all changes which resulted in lowered sperm intracellular pH, including lowered sea‐water pH, inclusion of the divalent ion chelator EGTA, addition of dithiothreitol, or removal of sea‐water Na+, enhanced sperm viability for periods of up to a week. These conditions caused decreased cell motility and elevated ATP concentrations, and prevented the acrosome reaction. Conversely, changes which increased the intracellular pH, decreased sperm ATP concentrations, or induced the acrosome reaction, reduced sperm viability. A single medium, high sea‐water K+ concentrations (greater than 100 mM), provided an exception to these general trends. At elevated K+ concentrations sperm were quiescent but became completely infertile. These data show that sperm fertilizing capacity is generally extended by maintenance of the sperm in an inactive state, and the results suggest that decreased cellular energy levels contribute to decreased fertility.

Molecular Alterations in Gamete Surfaces During Fertilization and Early Development

Publisher Summary This chapter discusses the molecular alterations in gamete surfaces during fertilization and early development. The early events of fertilization involve many changes at the surfaces of both gametes. The acrosome reaction of sperm is an absolute requirement for fertilization. The gametes perform activities characteristic of many specialized cell types. The movement of ions in the sperm as it undergoes the acrosome reaction is reminiscent of phenomena in excitable cells, such as muscle and nerve. The early changes that occur in the sperm, with an apparent rapid change in the membrane permeability to several ions, suggest that one of the first triggering mechanisms may involve alteration in the membrane potential of the cell. Upon fusing with the egg, the sperm membrane does not diffuse rapidly, as would be expected from studies of other cells, but rather exists as a patch throughout early development. The egg responds to the sperm by altering its surface, both by inserting some 10,000 cortical granules into the plasma membrane and by having a concomitant, and perhaps related, increase in the rigidity of the cortical zone. The egg additionally changes the properties of its glycocalyx, in assembling a complex fertilization membrane.

Ion measurements in sea urchin sperm.

Publisher Summary This chapter discusses methods used to measure ion changes important to activation of sea urchin sperm motility and the acrosome reaction. The methods used to measure pH i , ion fluxes, and membrane potentials in sea urchin sperm are described. Methods available for measurement of pH i include uptake of radiolabeled weak acids and bases, 31 P-nuclear magnetic resonance, and incorporation of molecular probes such as carboxyfluorescein that undergo spectral or fluorescent shifts with changes in pH i . Simple ionic manipulation, regulation of pH i , and [Ca 2+ ] are of fundamental importance to sperm physiology. pH i is discussed as it is an important regulatory component of sperm motility and the acrosome reaction. pH i changes in sea urchin sperm correlate with or are linked with other cation movements also. In particular, a Na + /H + exchange is a component of the sperm plasma membrane that can either raise or lower the pH i . Induction of the acrosome reaction results in multiple changes in the sperm cation composition. Na + and Ca 2+ enter the sperm and K + (and H + ) exit. Of these Ca 2+ entry is a crucial requirement for the acrosome reaction regardless of the method used to initiate it.

The Behavior of Sperm Before Fertilization

We know from a host of morphological studies that the sperm is linearly differentiated, with its acrosome, head, mitochondria and flagella spaced along the length of the cell. This allows not only for the cell’s interaction with its environment but also for fusion with an egg of the appropriate species to initiate development of a new organism. The sperm has a limited set of cellular functions; it swims to, or is transported to an egg and fuses with the egg plasma membrane by exposing through exocytosis the specialized contents of the acrosomal granule. In mammals these events also include capacitation. Sperm motility in all species results from flagellar movement in which the chemical energy of ATP is transformed into motion by the dynein ATPase. In echinoderms, the acrosome reaction consists of exocytosis of the acrosomal granule and polymerization of actin into the acrosomal rod.