Motonori Hoshi

Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzi: effects of protease inhibitors.

Abstract Effects of 15 proteinase inhibitors and an inhibitor against aminopeptidases on fertilization of the solitary ascidian, Halocynthia roretzi were studied in search of lysins. Fertilization of intact eggs was blocked by three trypsin inhibitors, leupeptin, antipain, and soybean trypsin inhibitor, and by two chymotrypsin inhibitors, chymostatin and potato proteinase inhibitor I. On the other hand, the fertilization of naked eggs was not blocked at all by leupeptin and was only partially blocked by chymostatin at the concentrations sufficient for blocking that of intact eggs. This indicates that spermatozoa utilize trypsin-like and chymotrypsin-like proteinases probably as lysins for penetrating through the chorion. The chymotrypsin-like activity appears to be also required for some step besides sperm penetration through the egg investments.

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca2+ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski‐microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction‐inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca2+.

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.

Separation of the sperm agglutinin and the acrosome reaction-inducing substance in egg jelly of starfish.

The egg jelly of the starfish Asterias amurensis was separated into the fractions J1, J2, and J3 on a Sephadex G-100 column. The Jl fraction induced the acrosome reaction and J2 induced sperm agglutination. Chemical analysis and chromatography revealed that sperm agglutinin is similar to asterosaponin A.

Arylsulfatase of sea urchin sperm: 2. Arylsulfatase as a lysin of sea urchins☆

Abstract An arylsulfatase is defined as a lysin of sea urchin sperm from the following evidences. (1) The activity is detected in the sperm of all the sea urchins investigated, and the activity is partially liberated from the cells after the acrosome reaction ( Moriya and Hoshi, 1979 ). (2) Fertilization is completely inhibited in the presence of 40 m Mp -nitrophenyl sulfate, which is an artificial substrate of arylsulfatase, but is not inhibited by p -nitrophenyl phosphate at the same concentration. (3) The inhibitory effect of p -nitrophenyl sulfate on fertilization is remarkably diminished by pretreatment of eggs with arylsulfatase before insemination. (4) Sperm arylsulfatase as well as limpet arylsulfatase appear to digest the vitelline coat and jelly coat.

Characterization and partial purification of arylsulfatase from the seminal plasma of the sea urchin, Strongylocentrotus intermedius

Abstract Arylsulfatase from seminal plasma of the sea urchin, Strongylocentrotus intermedius, was characterized with a crude enzyme preparation using p-nitrophenyl sulfate as the substrate. The enzyme which exists in soluble form, requires Ca2+ for the expression of full activity and calcium may be replaced by Ba2+, but only partially by Mg2+ and Sr2+. EDTA, heavy metals (Zn2+, Cu2+, Fe3+), and divalent anions including sulfate reduce the enzyme activity, however the enzyme is not so sensitive to sulfhydryl reagents. The inhibition with divalent anions is of a competitive nature. The optimum pH is 6.8 in 10 m m Tris-maleate buffer, but it shifts to 8.0 if the concentration of the buffer is increased to 100 m m . The enzyme hydrolyzes ascorbate-2-sulfate in addition to p-nitrophenyl sulfate, with Km values of 0.53 and 0.50 m m for p-nitrophenyl sulfate and ascorbate-2-sulfate, respectively. Cerebroside sulfate, seminolipid, cholesteryl sulfate, and proteochondroitin sulfate are not hydrolyzed appreciably by the enzyme. Although the enzyme is fairly stable at −20 °C, it is rather labile at 0 °C. The enzyme was partially purified by ammonium sulfate precipitation and successive chromatographies on Ultrogel AcA 34 and DEAE columns. At the final step, the enzyme was purified 47-fold, however, some heterogeneity on electrophoresis was evident.

EXOGASTRULATION INDUCED BY CHILLING IN SEA URCHIN LARVAE

Exogastrulation is induced by chilling in several species of sea urchins, including Strongylocentrotus intermedius, Strongylocentrotus nudus, Pseudocentrotus depressus and Anthocidaris crassispina, but not in Hemicentrotus pulcherrimus. When early gastrulae are raised at low temperatures, no pseudopodia of secondary mesenchyme cells are formed, but the invagination of the endodermal plate occurs normally. When gastrulae in later stage having pseudopodia are chilled, the pseudopodia withdraw and the archenteron begins to retract, resulting in exogastrulation. The exogastrulae are also induced when the larvae are raised in the presence of colchicine, vinblastine, cytochalasin B or cytochalasin C. The formation of exogastrula induced by chilling is presumed to be caused by the depolymerization of microtubules in the secondary mesenchyme cells and their pseudopodia. The fully invaginated archenteron of the late gastrula, even when it is chilled, remains within the blastocoel and does not evaginate.

Exogastrulation induced by heavy water in sea urchin larvae

Abstract Replacement of H 2 O with D, O in seawater causes exogastrulation in larvae of the sea urchins, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius and S. nudus . When larvae at any stages before mesenchymal blastula stage are transferred to 40% D 2 O-seawater all of them develop gradually to exogastrulae and finally up to plutei with evaginated archenterons. Effects of D 2 O are partly reversible at limited steps of the way to exogastrulation. Fertilisation and cleavage are not affected appreciably by D 2 O (50% or less) except for the delay of cleavage.

Arylsulfatase of sea urchin sperm—Distribution of arylsulfatase in the gonads and gametes of echinoderms

1. Fairly high activities of arylsulfatase are found in the sperm and mature testes of all the sea urchins studied; Strongylocentrotus intermedius, Strongylocentrotus nudus, Hemicentrotus pulcherrimus and Anthocidaris crassispina, whereas the activities in the ovaries and eggs of these animals are low. 2. Neither the sand dollar, Clypeaster japonicus nor the starfishes, Asterias amurensis and Asterina pectinifera prove to have considerable activities of the enzyme in their gonads and gametes. 3. Most of the activity of arylsulfatase in the sperm of S. intermedius is found in the seminal plasma, but the significant activity is bound to the spermatozoa. 4. Part, if not all, of the spermatozoa-borne arylsulfatase is suggested to exist on the surface of spermatozoa or in the acrosome or both. 5. The ubiquitous distribution of sperm arylsulfatase in sea urchins on the contrary to its absence in starfish or sand dollar is discussed in connection with the penetration of sperm through egg investments.