Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.     

Acrosome Reaction-Inducing Substance Purified from the Egg Jelly Inhibits the Jelly-Induced Acrosome Reaction in Starfish: An Apparent Contradiction

Previous studies indicated that two components of the egg jelly are required for induction of the acrosome reaction in starfish: a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) called Co-ARIS. In the present study the sites of action of ARIS and Co-ARIS and their temporal relationships were examined. When sperm had been treated for a few minutes with ARIS, or a crude preparation of Co-ARIS (Fraction M₈), or inadequate amounts of jelly, or sufficient jelly in low Ca²⁺ sea water, they did not undergo the acrosome reaction when the deficiencies were corrected. Moreover, they became nonresponsive to the jelly. Pronase digest of ARIS (P-ARIS) but not of Fraction M₈ retained this capacity. A steroidal saponin purified as Co-ARIS did not have this capacity. This suggests the presence of a third jelly component, probably an oligopeptide(s), participating in induction of the acrosome reaction. Activation of Ca²⁺ -uptake seems to be at least one, if not the only, action site of ARIS and Co-ARIS, because ARIS, P-ARIS, and Fraction M₈ inhibited jelly-induced Ca²⁺ -uptake by sperm, and because the calcium ionophore A23187 by-passed the blockage by these components of the jelly-induced acrosome reaction.     

Pretreatment effects of jelly components on the sperm acrosome reaction and histone degradation in the starfish, Asterina pectinifera.

Acrosome reaction (AR) and histone degradation (HD) of Asterina pectinifera sperm are induced by co-operation of ARIS and a diffusible fraction (M8) of egg jelly. Once sperm are treated with ARIS or M8 separately for several minutes, they do not undergo the AR in response to the egg jelly. Preincubation of sperm with M8 at 0 degrees C is not effective to block the jelly-induced AR whereas inhibitory effects of ARIS remain at 0 degrees C. Jelly-induced HD is inhibited by pretreatment of sperm with ARIS but is not affected by the incubation with M8. The blockage of the jelly-induced reactions, both AR and HD, by ARIS- or M8-pretreatment can be bypassed by ionophores, A23187 and monensin.     

Purification of Co-ARIS, a Cofactor for Acrosome Reaction-Inducing Substance, from the Egg Jelly of Starfish

In the starfish, Asterias amurensis , the acrosome reaction-inducing capacity of the egg jelly has been attributed to two components in the egg jelly: a sulfated glycoprotein (ARIS) and an unidentified low-molecular weight substance (Co-ARIS). In the process of purification of Co-ARIS, we found that Co-ARIS is not a single chemical entity but a series of closely related molecules. Co-ARIS was first group-separated by using octadecylsilane and anion-exchanger. Three major Co-ARIS' (I, II and III) were then purified by successive chromatographies using octadecylsilane and silica gel to the homogeneity in thin layer chromatography. Each of purified Co-ARIS', together with ARIS or the Pronase-digest of ARIS, induced the acrosome reaction at high ratios in normal seawater.     

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.     

Treatment of Starfish Sperm with Egg Jelly Induces the Degradation of Histones

When spermatozoa of Asterina pectinifera are treated with a solution of homologous egg jelly, besides undergoing the acrosome reaction, they begin to degrade their histones gradually. The degradation is most prominent with histone H1, almost 75% of which is degraded within one hour at 20°C. The jelly-induced histone degradation, like the acrosome reaction, requires external Ca²⁺, prefers high pHs and is susceptible to Ca²⁺-channel antagonists such as verapamil and diltiazem. Histone degradation is also induced by nigericin as well as monensin in normal seawater, but not in Ca²⁺-free seawater. Calcium ionophore A23187, that greatly facilitates the monensin-induced histone degradation, also induces histone degradation by itself, slightly in normal seawater and markedly in Ca²⁺-enriched seawater. Concanavalin A inhibits the jelly-induced histone degradation but not the jelly-induced acrosome reaction. These results suggest that egg jelly induces the histone degradation by enhancing Ca²⁺-influx via a Ca²⁺-channel(s) and by increasing cytoplasmic pH, through a pathway which is closely related to, but not entirely the same as, the one leading to the acrosome reaction.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis II. Purification and Characterization of Acrosome Reaction-Inducing Substance

The acrosome reaction-inducing substance (ARIS) was purified from egg jelly of the starfish, Asterias amurensis. The purification procedure included elimination of neutral glycoproteins from the ARIS fraction by isoelectric pointprecipitation and subsequent gel filtrations on Sephadex G–50 and Bio-Gel A-50m columns. The final preparation of ARIS was homogeneous as judged by cellulose acetate electrophoresis of ARIS and by ion-exchange chromatography on DEAE-Sephadex A–25 of S-carboxymethylated ARIS. ARIS is a very large, sulfated glycoprotein containing fucose, galactose, galactosamine and glucosamine as sugar components. It requires diffusible cofactor (Co-ARIS) for full biological activity. A Pronase digest of ARIS retained its capacity to induce the acrosome reaction when Co-ARIS was added to the bioassay system. The physiological significance of the carbohydrate moiety of ARIS is discussed.     

Novel conserved structural domains of acrosome reaction-inducing substance are widespread in invertebrates

In the starfish Asterias amurensis, acrosome reaction inducing substance (ARIS) is the main factor responsible for allowing sperm to recognize the egg jelly and begin the acrosome reaction (AR). ARIS is a large proteoglycan-like molecule, and its pentasaccharide repeat, Fragment 1 (Fr. 1), is responsible for inducing AR. Here, we investigated the primary structure of ARIS for the first time in order to improve our understanding of its functionality. Electrophoretic analysis revealed that ARIS is a complex of three proteins, all of which are modified by the Fr. 1 sugar chain. Sequencing indicated that there are two novel, conserved domains in all three ARIS proteins: ARIS N-terminus (AR-N) and ARIS C-terminus (AR-C) domains. We also found that other echinoderms possess ARIS proteins that are capable of inducing the AR for homologous sperm, indicating that ARIS proteins may be a ubiquitous component for echinoderm fertilization. Moreover, we identified ARIS-like genes from Ctenophora to Protochordata.     

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.     

Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca²⁺ and intracellular pH (pH_i). Since the two jelly components essential for inducing the acrosome reaction, ARIS and Co-ARIS, were shown to activate Ca-channels (accompanying paper), we examined the jelly components to determine which was responsible for the pH_i-increase using 9-aminoacridine as a probe of pH_i. This paper presents evidence that an oligopeptide(s) is responsible for the pH_i-increase. The pH_i of swimming sperm is 7.4-7.5. Within 20 sec after the addition of jelly, their pH_i increased rapidly by 0.06 pH unit, then decreased by 0.2–0.3 pH unit, and reached a plateau level within 3 min. Similar changes in pH_i were observed on addition of a Pronase digest of ARIS (P-ARIS) and a diffusible fraction of jelly (Fraction M₈) together. Fraction M₈, but not ARIS or Co-ARIS increased the pH_i, and activated sperm respiration in sea water at pH 6.5. The two activities of Fraction M₈ depended upon Na⁺ but not Ca²⁺, and were susceptible to Pronase digestion. Fraction M₈ is also known to enhance induction of the acrosome reaction by the Ca-ionophore A23187. These results suggest that the egg jelly contains a peptide(s) that is not obligatory for the acrosome reaction but facilitates the reaction by increasing the pH_i of the sperm. The significance of the pH_i-increase upon the acrosome reaction is discussed.     

Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca2+ and following acrosome reaction in starfish spermatozoa

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.     

NMDA‐type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm

The N‐methyl d ‐aspartate type glutamate receptor (NMDAR) is a ligand‐gated cation channel that causes Ca²⁺ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA‐seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg‐jelly substances along with acrosome reaction‐inducing substance (ARIS) and sperm motility‐initiating substance (SMIS). In the egg‐jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg²⁺, which blocks Ca²⁺ influx through gated NMDARs, was removed from the ST. In addition, the ARIS‐induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg²⁺ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.     

PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish

The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.     

Low-Na+Seawater Induces the Acrosome Reaction and Histone Degradation of Starfish Sperm in the Absence of Egg Jelly

Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera . Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na⁺-free seawater. It required external Ca²⁺ much less than the jelly-induced one in normal seawater, and was not susceptible to Ca²⁺-channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca²⁺-free seawater, they did not undergo the histone degradation even after subsequent addition of Ca²⁺, but Na⁺-free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10–30 mM Na⁺ in a Ca²⁺-dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low-Na⁺ seawater. Increasing K⁺ inhibited both pHi changes and the acrosome reaction induced by low-Na⁺ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.     

A novel saccharide structure, Xyl 1----3 Gal 1----(SO3-)3,4 Fuc----, is present in acrosome reaction-inducing substance of the starfish, Asterias amurensis.

The jelly coat of echinoderm eggs contains a glycoconjugate, acrosome reaction-inducing substance (ARIS), that is essential for triggering the acrosome reaction in homologous spermatozoa. In the starfish, Asterias amurensis, ARIS is a sulfated glycoprotein of an apparent molecular size of greater than 10(7). Since its biological activity is dependent mostly on its sugar moiety, oligosaccharides liberated by hydrolysis with 10 mM H2SO4 for 60 min at 100 degrees C from pronase digests of ARIS (P-ARIS) were chemically analyzed. The main oligosaccharide purified by high-performance anion-exchange chromatography was determined to be Xyl1----3Gal1----(SO3-)3,4Fuc by compositional analysis and FAB mass spectrometry. This structure indicates that ARIS possesses a novel saccharide chain having sulfated fucose as an internal residue.     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

Correlation Between the Molecular Structure and the Biological Activity of Co-ARIS, a Cofactor for Acrosome Reaction-Inducing Substance

Two components in the egg jelly are required for inducing the acrosome reaction in starfish; a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and its cofactor called Co-ARIS. Three distinct molecules were isolated as the major Co-ARIS' and designated as Co-ARIS' I, II and III. Structural analysis of Co-ARIS' revealed that they are steroidal saponins comprising a sulfated steroid and a pentasaccharide chain. Co-ARIS' I and II differ only in the steroidal side chain. In the presence of ARIS, each Co-ARIS induced the acrosome reaction with a maximal effect at 100–200 μM (Co-ARIS I) or 25–50 μM (Co-ARIS' II and III). Mixtures of Co-ARIS' I, II and III were more effective than the individuals. The activity of Co-ARIS was considerably reduced by solvolytic desulfation but was not affected at all by periodate oxidation. Reduction by NaBH₄ decreased the activity of Co-ARIS I and enhanced that of Co-ARIS II. Treatment of Co-ARIS III with NaBH₄ did not affect the activity as anticipated from its structure. These results suggest that the sulfate moiety and the side chain of steroid are important for the activity of Co-ARIS. The saccharide chain, however, seems not necessarily to be strictly specified for the activity.     

Monoclonal antibodies to a membrane glycoprotein induce the phosphorylation of histone H1 in sea urchin spermatozoa

Two groups of mAbs reacting with external domains of a major sea urchin sperm membrane glycoprotein of 210 kD were isolated. Previous studies have shown that group I mAbs inhibit the acrosome reaction induced by egg jelly and also cause large increases in intracellular Ca2+ [( Ca2+]i). Group II mAbs, at comparable levels of cell surface binding, neither inhibit the egg jelly-induced acrosome reaction nor cause increases in [Ca2+]i. In this paper, we investigate the ability of these mAbs to induce the cAMP-dependent phosphorylation of sperm histone H1. Group I mAbs induce H1 phosphorylation to the same level and on the same peptide, as occurs upon treatment of sperm with egg jelly. These mAbs also activate adenylate cyclase to the same extent as egg jelly. Group II mAbs do not induce H1 phosphorylation and are only poor activators of adenylate cyclase. Group I mAbs compete with each other, but not with group II mAbs, for binding to the cell surface. These data indicate that the activation of adenylate cyclase is an initial event in the pathway leading from the binding of mAbs to a specific domain of the 210-kD protein at the cell surface, to the discrete phosphorylation of histone H1 in highly condensed sperm chromatin. The domain on the 210-kD protein recognized by group I mAbs plays a critical role in signal transduction during the early events of fertilization.     

Phosphorylation of sperm histone H1 is induced by the egg jelly layer in the sea urchin Strongylocentrotus purpuratus

Phosphorylation of histone H1 occurs when spermatozoa of the sea urchin Strongylocentrotus purpuratus are treated with the macromolecular fraction of solubilized egg jelly. Phosphorylation is on serine residues in the N-terminal fragment of H1 bisected with N-bromosuccinimide. Phosphorylation is maximal by 4-8 min and dependent on Ca2+, but independent of Na+ or increased intracellular pH. Phosphorylation of H1 can be dissociated from the induction of the acrosome reaction. Only a fraction of the H1 molecules become phosphorylated upon treatment of sperm with egg jelly. The amount of phosphate per mole of H1 increases from 0.15 moles before jelly treatment to 0.46 moles after maximal phosphorylation. Phosphorylation of H1 occurs in a cAMP-dependent manner as indicated by the ability of the phosphodiesterase inhibitors IBMX and SQ20009 to induce H1 phosphorylation. This phosphorylation reaction can be blocked by digesting the sperm surface with Pronase, or preincubation of sperm in wheat germ agglutinin, showing that a ligand in egg jelly must interact with a sperm surface receptor to activate the kinase phosphorylating H1.