Physiological inducers of the acrosome reaction

This article reviews recent studies on physiological inducers of the acrosome reaction in starfish. Upon encountering the jelly coat of eggs, starfish sperm undergo the acrosome reaction in response to a cooperation of three jelly components: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of steroidal saponins named Co-ARIS, and an oligopeptide presumably having an activity to increase the intracellular pH of sperm. ARIS induces the acrosome reaction in high Ca²⁺ or high pH sea water. In normal sea water, both ARIS and Co-ARIS are required for the induction. In addition to ARIS and Co-ARIS, a third jelly component, the oligopeptide, is necessary to mimic the full capacity of the jelly coat to induce the acrosome reaction. ARIS and Co-ARIS cooperatively increase the intracellular Ca²⁺ by stimulating Ca²⁺ channels, while the oligopeptide increases the intracellular pH by stimulating Na⁺/H⁺ exchange systems. When sperm meet the eggs, both changes are simultaneously achieved in them and thus they undergo the acrosome reaction.     

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.     

Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis, and Lytechinus variegatus, were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.     

Activation of Ca2+channels during the acrosome reaction of sea urchin sperm is inhibited by inhibitors of chymotrypsin-like proteases

Probable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p-nitrophenyl-p′-guanidinobenzoate (NPGB), a serine protease inhibitor, and N-benzoyl-L-tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly-induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly-induced acrosome reaction, intraccllular Ca²⁺ concentration ([Ca²⁺]_i) and pH (pH_i) were measured with fura-2 and 2′,7′-bis (carboxy-ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca²⁺]_i which was depressed by BTEE. Egg jelly also caused a transient rise of pH_i, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca²⁺]_i. The rise of pHj was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin-like protease of sea urchin sperm activates verapamil-sensitive Ca²⁺ channels, which take part in the acrosome reaction.     

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm.

We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.     

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

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.     

High pH-induced acrosome reaction and Ca 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.     

Egg water from the amphibian Bufo arenarum induces capacitation-like changes in homologous spermatozoa

Mammalian sperm acquire fertilizing capacity after residing in the female tract, where physiological changes named capacitation take place. In animals with external fertilization as amphibians, gamete interactions are first established between sperm and molecules of the egg jelly coat released into the medium. Since dejellied oocytes are not normally fertilized, the aim of this study was to determine if the jelly coat of the toad Bufo arenarum promotes a “capacitating” activity on homologous sperm. We found that sperm incubation in diffusible substances of the jelly coat (egg water) for 90-180 s is sufficient to render sperm transiently capable of fertilizing dejellied oocytes. The fertilizing state was correlated with an increase of protein tyrosine phosphorylation and a decrease of sperm cholesterol content. Inhibition of either the increase in tyrosine phosphorylation or cholesterol efflux affected the acquisition of fertilizing capacity. Phosphorylation and fertilization could be promoted with NaHCO₃ and also by addition of beta cyclodextrin. Moreover, sperm could gain the ability to fertilize dejellied oocytes in the presence of these compounds. These data indicate that sperm should undergo a series of molecular changes to gain fertilizing capacity; these changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction.     

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.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen Peroxide-mediated Oxidation of Indole-3-acetic Acid by Tomato Peroxidase and Molecular Oxygen

The oxidation of indole-3-acetic acid by anionic tomato peroxidase was found to be negligible unless reaction mixtures were supplemented with H₂O₂. The addition of H₂O₂ to reaction mixtures initiated a period of rapid indole-3-acetic acid oxidation and O₂ uptake; this phase ended and O₂ uptake fell to a low level when the H₂O₂ was exhausted. The stoichiometry of the reaction, which is highly dependent on enzyme concentration and pH, suggests that H₂O₂ initiates a sequence of reactions in which indole-3-acetic acid is oxidized.     

Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin

Recent evidence suggests roles for egg derived hydrogen peroxide (H₂O₂) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H₂O₂ during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H₂O₂ resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H₂O₂-treated sperm. Phenylhydrazine acted to protect sperm fertility from H₂O₂, while 3-amino-1,2,4-triazole increased the adverse effect of H₂O₂. Simultaneous addition of both inhibitors to sperm incubated in H₂O₂ gave an intermediate value of sperm fertility. These data indicate that (1) H₂O₂ generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H₂O₂ reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H₂O₂. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.     

A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion

The classical isoagglutination of sea urchin sperm by egg jelly is not an agglutination of cells, as proposed by the fertilizin-antifertilizin hypothesis. Sperm motility is required to obtain the isoagglutination of Strongylocentrotus purpuratus sperm, and the sperm do not adhere to each other in the isoagglutination clusters, which cannot be fixed for microscopy and which disperse rapidly into individual cells when sperm motility is inhibited. These observations suggest that isoagglutination is the swarming of freely moving sperm to a common focus and is quite distinct from the agglutination of sperm by known crosslinking agents (antibodies or lectins).A previously unrecognized form of sperm agglutination is described which follows induction of an acrosome reaction by egg jelly, ammonia, or the ionophore A23187 in a suspension of sea urchin or sand dollar sperm. The sperm form rosettes of up to 100 cells in which the newly extended acrosomal processes adhere to each other. Rosettes can form containing sperm of different species, in which the acrosomal processes adhere without species preference.As observed by transmission electron microscopy, the acrosomal process of Lytechinus pictus sperm consists of an acrosomal tubule covered by a sheath of extracellular material. Rosette formation results from attachment between the extracellular materials of adjacent sperm.Less frequently, the acrosomal process of one sperm adheres to the midpiece of another by fusion of the acrosomal tubule and midpiece plasma membranes.     

When do sperm of the sea urchin,Pseudocentrotus depressus,undergo the acrosome reaction at fertilization?

Jelly coats of the sea urchin, Pseudocentrotus depressus, were stripped off the eggs, and the eggs were “inseminated.” After penetration through the isolated jelly hull, sperm swarmed in the cavity previously occupied by the egg. Electron microscopic examination could not detect any sperm with reacted acrosome. Observation was also made of the sperm penetrating through the intact jelly coat-egg complex. Although a number of sperm were examined in ultrathin sections, only those attached to the vitelline layer had undergone the acrosome reaction; those sperm embedded in jelly but not attached to the vitelline layer had not undergone the acrosome reaction. The sequence of events in fertilization of this species and of other echinoids is discussed.     

Induction of the Acrosome Reaction in Starfish

In the starfish, Asterias amurensis , at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) named Co-ARIS. The following evidence suggested that ARIS and Co-ARIS cooperatively activate CA-channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P-ARIS) and Co-ARIS, either as a pure or a crude preparation (Fraction M₈), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P- ARIS alone induced the acrosome reaction fully in high Ca²⁺ sea water and markedly at high pHs, whereas Fraction M₈ alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca²⁺ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca²⁺ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M₈. Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M₈ caused abrupt Ca²⁺ -uptake by the sperm. The Ca-channel blockers verapamil and diltiazem inhibited the jelly-induced acrosome reaction.     

The Participation of Speract in the Acrosome Reaction of Hemicentrotus pulcherrimus

A speract-free macromolecular fraction was prepared from the egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration and tested for ability to induce the acrosome reaction in H. pulcherrimus spermatozoa with or without exogenously added synthetic speract. The macromolecular fraction without speract showed only about half the activity of the original unfractionated jelly for induction of the acrosome reaction. The rates of the acrosome reaction induced by the fraction with speract were comparable with those induced by the unfractionated jelly at all pHs tested. Speract itself, however, did not induce the acrosome reaction in the absence of the macromolecular fraction of jelly. The acrosome reaction was associated with incerase of the cyclic AMP concentration in sperm cells, the extent of incerase depending on the concentration of the macromolecular fraction. Addition of speract to the fraction enhanced both induction of the acrosome reaction and increase in the cyclic AMP concentration induced by the fraction. These results suggest that a major factor(s) responsible for the acrosome reaction is a macromolecular component(s) of the jelly and that speract promotes the reaction as a co-factor.     

Egg water from the amphibian Bufo arenarum modulates the ability of homologous sperm to undergo the acrosome reaction in the presence of the vitelline envelope

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed "egg water" (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca(2+) stores (induced by thapsigargin) promoted [Ca(2+)](i) rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca(2+) chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni(2+) and mibefradil prevented [Ca(2+)](i) rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca(2+) channel.     

Oxidation of dehydroascorbic acid and 2,3-diketogulonate under plant apoplastic conditions

The rate of l-ascorbate catabolism in plants often correlates positively with the rate of cell expansion. The reason for this correlation is difficult to explore because of our incomplete knowledge of ascorbate catabolism pathways. These involve enzymic and/or non-enzymic oxidation to dehydroascorbic acid (DHA), which may then be hydrolysed to 2,3-diketogulonate (DKG). Both DHA and DKG were susceptible to further oxidation under conditions of pH and H₂O₂ concentration comparable with the plant apoplast. The kinetics of their oxidation and the identity of some of the products have been investigated here. DHA, whether added in pure form or generated in situ by ascorbate oxidation, was oxidised non-enzymically to yield, almost simultaneously, a monoanion (cyclic-oxalyl-threonate; cOxT) and a dianion (oxalyl-threonate; OxT). The monoanion was resistant to periodate oxidation, showing that it was not oxalic threonic anhydride. The OxT population was shown to be an interconverting mixture of 3-OxT and 4-OxT, differing in pK_a. The 3-OxT appeared to be formed earlier than 4-OxT, but the latter predominated at equilibrium. DKG was oxidised by H₂O₂ to two partially characterised products, one of which was itself further oxidised by H₂O₂ to yield threonate. The possible occurrence of these reactions in the apoplast in vivo and the biological roles of vitamin C catabolites are discussed.     

Jelly coat and induction of the acrosome reaction in echinoid sperm

In a previous study we established that sperm from four species of echinoids differ in their specificity for induction of the acrosome reaction by heterotypic jelly coat and we presented evidence that there were only small compositional differences in the active component of the jelly coat, a polysaccharide composed of fucose sulfate units. In the current report we present additional studies related to the species specificity of jelly coat with respect to Ca²⁺ uptake (or exchange), which occurs concomitantly with the acrosome reaction, and activation of phospholipase activity, which appears to occur subsequent to the acrosome reaction. The specificity of jelly coat in inducing these processes is the same as that previously observed in induction of the acrosome reaction. Binding of jelly coat to sperm has been demonstrated, and has been shown to be species specific. This finding raises the possibility that a receptor for jelly coat exists on the surface of the sperm. Finally, based on chemical and physical-chemical studies, evidence is presented that establishes that, despite compositional similarities, the fucose sulfate polysaccharides from the four species of eggs differ in structure.