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.     

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.     

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.     

罗氏海盘车(Asterias rollestoni)中几种化合物的提取分离和结构鉴定

罗氏海盘车的95％EtOH提取物用大孔吸附树脂柱、凝胶柱和硅胶柱进行反复分离得到4个化合物,用NMR、MS和IR分析技术分别鉴定为苯丙氨酸、2′-脱氧胸腺嘧啶核苷、2′脱氧次黄嘌呤核苷-5′-单磷酸盐和5-甲氧基-1-十三醇。     

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.     

Separation of Ovarian Asterosaponins in the Starfish Asterias amurensis

Both the enantiomers of (Z)-cis-9,10-epoxy-6-heneicosene (2), a pheromone component of Phragmatobia fuliginosa L, were synthesized by employing the Sharpless asymmetric epoxidation as the key step.     

Structure of Ovarian Asterosaponin-1 in the Starfish Asterias amurensis

Cultured crown gall cells of Catharanthus roseus Don (Vinca rosea L.) was found to contain brassinosteroids. These were identified as brassinolide and castasterone by GC/MS. This is the first conclusive identification of endogenous brassinosteroids in cultured cells.     

Properties of Asterosaponin B Isolated from a Starfish,Asterias amurensis

Succeeding to asterosaponin A, the second saponin component has been isolated from a starfish (Asterias amurensis) and designated asterosaponin B. It contains a conjugated ketone and one molecule of sulfuric acid as the sodium salt. The sugar moiety consists of two molecules of d-quinovose and one molecule each of D-fucose, d-xylose, and d-galactose, differing from that of asterosaponin A consisting of two molecules each of d-quinovose and d-fucose. On acid hydrolysis both asterosaponins A and B yielded the similar mixture of aglycon components. The two main components isolated were designated asterogenins I and II, respectively.     

Isolation and Characterization of Spawning Inhibitors in Ovary of the Starfish,Asterias amurensis

Biologically active substances which inhibit spawning of mature starfish, Asterias amurensis, have been isolated from ovaries of the same organisms and identified as asterosaponins A and B.     

Isolation and Identification of Spawning Inhibitors from Ovaries of the Starfish,Asterias amurensis

An extracellular alkaline proteinase produced by Candida lipolytica was purified through iso-propanol and ammonium sulfate precipitation, decolorization with DEAE-cellulose, gel filtration with Sephadex G–100 and ion-exchange chromatography on DEAE-Sephadex A–50. The optimum pH of its caseinolytic activity was 9.0, and this activity was completely inactivated with DFP but not with chelating reagents, PCMB, STI, TLCK, TPCK, or SSI. This enzyme also hydrolyzed salmin and synthetic esters, such as Bz. Arg. OEt, Bz. His. OMe, Tos. Lys. OMe or Ac. Tyr. OEt, and the optimum pH of its esterase activity was 8.0. The molecular weight of the enzyme was estimated to be about 30,000 by the gel filtration method. These facts indicated that this enzyme was distinguishable from other microbial alkaline proteinases so far studied.     

Ultrastructural Localization of Calcium in the Acrosome and Jelly Coat of Starfish Gametes

The potassium pyroantimonate technique was employed to localize calcium ultrastructurally on both male and female starfish gamete regions that first interact at fertilization. In the spermatozoon of Marthasterias glacialis , antimonate precipitates in the peripheral dense component of the acrosomal vesicle, while in the oocyte it precipitates in the jelly coat and beneath the oolemma. Calcium was identified in the precipitates by testing the chelator-sensitivity and by X-ray microanalysis of the precipitates.     

Changes in the Pattern of Intercellular Junctions during Early Embryogenesis of the Starfish, Asterias amurensis

Changes in the cellular adhesion pattern during the early embryogenesis of a starfish Asterias amurensis were examined using carboxyfluorescein (CF) dye as a probe. CF that was injected into one of the blastomeres at the 2- or 4-cell stage was in all cases restricted to the progeny cells of the CF-labelled blastomere. With the advancement of gastrulation, however, the injected dye was distributed not only to the progeny of the labelled blastomere, but also to cells that originated from non-injected blastomeres. At the beginning of mesenchyme cell release, the injected dye spread uniformly to most cells comprising the embryo. When one of the blastomeres situated in the vegetal hemisphere of an 8-cell embryo was labelled, the resulting embryo showed more intense fluorescence in the cells surrounding the archenteron than in the ectodermal layer, suggesting that the cells in ectodermal layer became associated more intimately or earlier than those surrounding the archenteron. Likewise, in double embryos formed by combining two denuded eggs, in which one egg had been labelled with CF, dye spread was observed when the ectodermal layer began to expand. The intercellular spread of CF dye in starfish embryo suggests that there is a dramatic change in the cellular adhesion pattern during the course of gastrulation.     

Effects of Oestradiol-17 β on the Ovaries of the Starfish Asterias rubens

Oocyte diameters and their frequency distribution, and various other data determined for the oestradiol-17 β treated female specimens of Asterias rubens proved to be significantly different from those for the control animals. The maturation index of the treated animals is 2.4, that of the control animals 1.4. Since the treated animals show a greater heterogeneity in development than the control animals, and because the diameter of the smallest oocytes is the same for both treated and control animals, a threshold size of the oocytes may be required before oocyte growth can be stimulated by oestradiol-17β, and before substances originating in the pyloric caeca are incorporated into the oocytes. Oestradiol-17β treatment caused a tenfold increase of the oestrone level in the ovaries, whereas a non-significant increase was observed in the pyloric caeca. This may indicate that in vivo oestradiol-17β is converted into oestrone in the ovaries but not in the pyloric caeca.     

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. I. WGA Affects the Ion Fluxes Associated with the Acrosome Reaction

The lectin wheat germ agglutinin (WGA) inhibited the egg jelly-induced acrosome reaction (AR) of sperm of the sea urchin, Strongylocentrotus intermedius . Fluorescein-conjugated WGA applied to sperm bound to the acrosomal region, to the midpiece, and to the tip of the flagellum. These effects were not observed in the presence of N-acetly-D-glucosamine. When the egg jelly was replaced by artificial AR inducers such as A23187 or nigericin, the AR was not inhibited by WGA. Results obtained using a Ca²⁺ indicator fura-2, a pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) and a membrane potential sensitive dye 3,3'-dipentyl 2,2'-dioxacarbocyanine [diO-C₅(3)] showed that WGA suppresses the egg jelly-induced influx of Ca²⁺ and slightly suppresses the efflux of H⁺ caused by the egg jelly, whereas the depolarization of the plasma membrane by the egg jelly is remarkably amplified by the treatment with WGA. These results suggest that WGA affects the regulatory system of the ion fluxes associated with the AR. The target protein of WGA (WGA-binding protein) was a membrane glycoprotein of 260 kD under non-reducing condition.     

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.     

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.     

Structure of Ovarian Asterosaponin-4, an Inhibitor of Spontaneous Oocyte Maturation from the Starfish Asterias amurensis

The ovary of the starfish Asterias amurensis contains various kinds of steroidal saponins. The structure of one of the major saponins, designated ovarian asterosaponin-4, was elucidated by several chemical modifications and spectroscopic analyses. The structure was formulated as 20α -hydroxy-6 α -O-{6-deoxy-β -D-glucopyranosyl(1→2)- β -D-glucopyranosyl(1→4)-[6-deoxy- β -D-glucopyranosyl (1→2)]- β -D-xylopyranosyl(1→3)-6-deoxy->β-D-gluco-pyranosyl}-3β-sulfo-oxy-5α -cholesta-9(11), 24-diene. Ovarian asterosaponin-4 at 125μg/ml inhibited spontaneous oocyte maturation of the starfish, and the inhibition could be overcome by adding 1 × 10?6M of 1-methyladenine, the maturation-inducing substance of the starfishes, to the oocyte.