Taei Matsui

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.

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 (pHi). 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 pHi‐increase using 9‐aminoacridine as a probe of pHi. This paper presents evidence that an oligopeptide(s) is responsible for the pHi‐increase. The pHi of swimming sperm is 7.4‐7.5. Within 20 sec after the addition of jelly, their pHi 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 pHi 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 pHi, 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 pHi of the sperm. The significance of the pHi‐increase upon the acrosome reaction is discussed.

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.

Induction of the Acrosome Reaction in Starfish

When starfish spermatozoa reach the jelly coat of homologous eggs, they immediately undergo the acrosome reaction that is a prerequisite for fertilization. Three organic components of the egg jelly are responsible for this phenomenon; namely, an extremely large sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of sulfated steroid saponins named Co-ARIS, and a sperm activating oligopeptide(s) (SAP). ARIS induces the acrosome reaction species-specifically in high Ca2+ or high pH sea water, but it requires Co-ARIS for the induction in normal sea water. The acrosome reaction induced by ARIS and Co-ARIS is not accompanied with a transient increase in the intracellular pH (pHi), which is generally accepted to be inevitable for triggering the acrosome reaction, and proceeds slower than the jelly-induced acrosome reaction. All the three, but nothing else, are required to reconstruct the acrosome reaction-inducing ability of the egg jelly.

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.

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.

Egg jelly components responsible for histone degradation and acrosome reaction in the starfish, Asterina pectinifera

In the starfish, Asterina pectinifera, egg jelly induces the degradation of sperm histones as well as the acrosome reaction. We have isolated histone degradation-inducing components from the egg jelly. The histone degradation and the acrosome reaction are induced by a co-operative action of ARIS, which is an extremely large, sulfated glycoprotein with diffusible substance(s) in the jelly. Co-ARIS I, a steroidal saponin of the jelly, is effective to induce both reactions in the presence of ARIS.

Maitotoxin, A Presumed Calcium Channel Activator, Induces the Acrosome Reaction in Mussel Spermatozoa

Maitotoxin, a presumed activator of the voltage‐sensitive calcium channel, induced the acrosome reaction in the mussel, Mytilus edulis at physiological pH and in the starfish, Asterias amurensis at pH 9.5. The induction of acrosome reaction by maitotoxin depended upon external Ca2+ and was inhibited by two types of calcium channel blockers; verapamil and diltiazem. These results suggest that the activation of the voltage‐sensitive calcium channel takes an important part in the initiation of acrosome reaction in Mytilus and other animals.

Anion Channel Blockers Inhibit the Acrosome Reaction of Echinoderm Sperm

Two types of anion channel blockers, SITS (4‐acetamide‐4‘‐isothiocyanostilbene‐2,2′‐disulfonic acid) and DIDS (4,4′‐diisothiocyanostilbene‐2, 2’‐disulfonic acid), inhibited jelly‐induced acrosorne reaction in starfish and sea urchin. In starfish sperm, both of the blockers reversibly inhibited the formation of acrosomal process but they had no effect on either the acrosomal exocytosis or acid release from the sperm. Complete acrosome reaction occurred even in Cl−‐ and SO42−‐free artificial seawater whereas HCO3−was required for the acrosomal exocytosis. Importance of anion transport in acrosome reaction is discussed.

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.