Rapid immunoassays for the acrosome reaction of sea urchin sperm utilizing antibody to bindin

Two assays employing rhodamine-conjugated and peroxidase-conjugated antibindin are presented. The assays are used to detect the occurrence of the acrosome reaction in sea urchin sperm. The assays are rapid and can be performed on large numbers of samples. The scoring of the reacted and unreacted cells can be done accurately at a rate of 60 cells/min.     

Antibody to a sperm surface glycoprotein inhibits the egg jelly-induced acrosome reaction of sea urchin sperm

When the surface of sea urchin (Strongylocentrotus purpuratus) sperm is radioiodinated, 75% of the protein-incorporated radioactivity is associated with two glycoproteins of M_r 84,000 (84K) 64,000 (64K) (Lopo and Vacquier 1980). Antibodies were prepared against these two components by separating a Triton X-100 extract of sperm on SDS-polyacrylamide gels, cutting out the band containing the glycoprotein and injecting the homogenized gel into rabbits. Both anti-84K and anti-64K sera agglutinate sperm. Light and EM immunoperoxidase localization show both antigens are distributed over the entire sperm surface. By the immunoperoxidase technique there is some degree of cross-reactivity of both antisera with sperm of other Strongylocentrotus species, but not with those of other genera. Living sperm incubated with anti-84K Fab fragments are completely inhibited from undergoing the egg jelly-induced acrosome reaction and fertilizing eggs. Anti-64K Fab fragments have no effect on the ability of the sperm to undergo the acrosome reaction or fertilize eggs. Sperm incubated in anti-84K or anti-64K Fab fragments undergo the acrosome reaction in response to the Ca²⁺ ionophore A23187, or when the extracellular pH is increased to 9.2 with NH₄OH, indicating that the inhibition of the egg jelly-induced acrosome reaction results from the binding of the anti-84K Fab to an external molecule involved in the initiation or propagation of the acrosome reaction. The 84K glycoprotein is the first sperm surface component identified that might have a role in the induction of the acrosome reaction.     

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.     

Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.     

Inhibition of sea urchin sperm acrosome reaction by antibodies directed against two sperm membrane proteins : Characterization and mechanism of action

The egg jelly-induced acrosome reaction is inhibited by polyclonal antibodies raised against either of two S. purpuratus sperm-membrane proteins, of M_r 80 and 210 kD. Although the two antigens used have dissimilar CNBr peptide maps, antisera produced against each of them cross-react with both proteins. Inhibition of the egg jelly-induced acrosome reaction by the antisera is bypassed by a combination of the ionophores monensin and A23187. This result, along with data showing that the antisera inhibit egg jelly-induced uptake of ⁴⁵Ca²⁺, suggests that the antisera may block both Ca²⁺ uptake and Na⁺/H⁺ exchange in the sperm. The acrosome reaction blockage appears to be caused by the same component of the polyclonal sera responsible for cross-reaction; consequently, these antisera cannot be used to determine whether one or both of the crossreacting proteins modulate a critical step in the acrosome reaction.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

Egg sialoglycans increase intracellular pH and potentiate the acrosome reaction of sea urchin sperm.

Sea urchin egg jelly (EJ) triggers sperm acrosome reaction (AR), an exocytotic event required for membrane fusion of the gametes. Purified fucose sulfate polymer (FSP) in EJ is one inducer of the AR. Binding of FSP to its receptor regulates opening of two distinct calcium channels and also elevates intracellular pH (pH(i)). EJ also contains sialic acid-rich glycans (sialoglycans (SG)) that were isolated by beta-elimination followed by DEAE chromatography. In the presence of limiting amounts of FSP, the SG fraction markedly potentiates the AR; however, by itself SG has no activity. The SG fraction increases the pH(i) of sperm without increasing intracellular Ca(2+). The SG-induced increase in pH(i) is not blocked by nifedipine or high K(+), whereas the FSP-induced pH(i) increase is sensitive to both these agents. Treatment of the SG fraction with neuraminidase or mild metaperiodate that specifically cleaves the glycerol side chain of sialic acid abolishes the AR potentiation and ability of SG to elevate pH(i). These data are the first to show that there are at least two pathways to induce sperm pH(i) increase and that egg surface sialic acid plays a role in triggering the sperm AR.     

Structural requirements for species-specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan

The sulfated fucan (SF) of egg jelly induces the acrosome reaction (AR) of sea urchin sperm. Strongylocentrotus franciscanus (Sf) SF is sulfated only at the 2-position. Strongylocentrotus purpuratus (Sp) has two SF isotypes, each one being female specific. One is rich in sulfate at both the 2- and 4-positionS (SF-1), and the other is rich in sulfate at the 4-position, but not the 2-position (SF-2). Sf SF is poor at inducing the AR of Sp sperm, presumably due to lack of 4-sulfation. Sp SF-1 is better at inducing the AR of Sf sperm than Sp SF-2, hypothetically due to increased 2-sulfation. Chemical oversulfation of Sf SF increases the percentage of AR of Sp sperm, showing that 4-sulfation is important for recognition of SF by Sp sperm. Chemically oversulfated Sp SF-2 is better at inducing the Sf sperm AR, presumably because of increased 2-sulfation. The species, Strongylocentrotus drobachiensis (Sd), has an SF-2 that is exclusively 2-sulfated (like Sf), except the glycosidic linkage in Sd is alpha(1-->4), whereas in Sf it is alpha(1-->3). Sd SF-2 does not induce the AR of Sf sperm, showing the strict requirement for the alpha(1-->3) linkage in recognition between Sf sperm and SF. Egg jelly from Echinometra lucunter (El) contains sulfated galactan (SG) which differs from Sf SF only in that the monosaccharide is L-galactose, not L-fucose. This SG and Sf SF are equally potent in inducing the AR of Sf sperm, showing that modification at C6 of L-fucose is not important for proper recognition between SF and Sf sperm receptors. This system permits study of the structural basis for recognition between sulfated polysaccharide and receptors controlling signal transduction pathways in animal cells.     

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((?)δ⁹ THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [ H]CP-55,940 to sperm, defined as total binding displaced by (?)δ⁹ THC, was saturable: K_D 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (?)δ⁹ THC, and (+)δ⁹ THC. The rank order of potency to inhibit binding of [ H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (?)δ⁹THC > (+)δ⁹THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley-Liss, Inc.     

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 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.     

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.     

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.     

Egg fucose sulfate polymer,sialoglycan, and speract all trigger the sea urchin sperm acrosome reaction

Macromolecules surrounding eggs induce the acrosome reaction (AR) of spermatozoa. In sea urchins, three egg jelly (EJ) molecules: a fucose sulfate polymer (FSP), a sialoglycan (SG), and speract mediate ionic fluxes triggering the AR. SG and speract are noninductive without FSP. Speract's role in AR induction is controversial. Here we show that speract potentiates the FSP-induced AR at pH 7.0, approximately 1 pH unit lower than natural seawater. At pH 7.0, a mixture of FSP, SG, and speract produces the intracellular pH increase necessary for maximum AR induction. Each EJ component may mediate a distinct intracellular pH control mechanism, and all three may function synergistically to increase the intracellular pH permitting AR induction. Speract peptides are an ancient family. Although important for activating cyclic nucleotide-mediated pathways in today's seawater of pH approximately 8, speract may have been more important in AR induction in the paleo-ocean of pH approximately 7.     

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.     

Mouse sperm antigens that participate in fertilization. IV. A monoclonal antibody prevents zona penetration by inhibition of the acrosome reaction

To investigate the molecular basis of gamete interaction in mammals, monoclonal antibodies (mAbs) have been generated by syngeneic immunization with mouse testis. Previous work has described two particular mAbs, M41 and M42, which localize indistinguishably to the plasma membrane overlying a restricted portion of the acrosome, but recognize different antigens. One of the mAbs, M42, inhibits mouse fertilization in vitro significantly, but only in the presence of the zona pellucida, whereas M41 has no apparent effect upon any assayable event in the fertilization process. The experiments described here were performed to identify the precise event of sperm-zona interaction (sperm-zona binding, induction of the acrosome reaction, or penetration through the zona) that is affected by M42 mAb. Capacitated mouse sperm binding to the zona pellucida was undiminished following pretreatment with M42 mAb, when compared to levels achieved using either no mAb- or to M41 mAb-treated control sperm. When the effect of mAbs on the zona-induced AR was examined, the percentage of acrosome reacted (AR) sperm at the zona surface increased with time, plateauing at approximately 90 min post-insemination, with 78% of the bound cells AR in the control and the M41 mAb-treated groups. M42-treated sperm never achieved greater than 23% AR cells over the 120-min interval assayed. To quantitate this effect, capacitated sperm were exposed to increasing concentrations of acid-solubilized zonae. Increased proportions of AR sperm were found in the control and M41 mAb-treated groups, up to a maximum of 70-76% AR cells with 8 or 12 zonae/microliter. In contrast, M42-treated sperm displayed only 21-28% AR cells over the entire range of zonae concentrations tested. An entirely different result emerged when acrosome reactions were induced with A23187: M42 was no longer able to prevent the AR. This ability of A23187 to override M42 mAb's inhibitory effect on the AR permitted specific examination of the possible effect of M42 mAb on sperm penetration through the zona pellucida. In the presence of A23187, zona penetration levels for M42 mAb-treated sperm were equivalent, both qualitatively and quantitatively, to control and to M41 mAb-treated sperm under the same conditions. It appears, therefore, that M42 mAb identifies a high molecular weight doublet (220-240 kDa) of mouse sperm that participates specifically in the induction of the sperm's acrosome reaction as it occurs under physiological conditions.