Isolation and purification of three egg-membrane lysins from sperm of marine invertebrate Megathura crenulata (giant keyhole limpet)

Heller, E.; Raftery, Michael A.

doi:10.1021/bi00745a013

Cited by 17 publications

(10 citation statements)

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“…Interaction of sperm and egg plasma membranes was not observed. This hypothesis of stepwise acrosomal action at more than one site is supported by our observations of the structure and changes of shape in the acrosome of R. pacifica, and by the occurrence of three eggmembrane lysins in the sperm of one molluscan species (Heller and Raftery 1973).…”

Section: Discussionsupporting

confidence: 73%

See 1 more Smart Citation

Ultrastructure and functional morphology of spermatozoa of Rossia pacifica (Cephalopoda, Decapoda)

Fields¹,

Thompson²

1976

Can. J. Zool.

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FIELDS, W. G., and K. A. THOMPSON. 1976. Ultrastructure and functional morphology of spermatozoa of Rossia pacifica (Cephalopoda, Decapoda). Can. J. Zool. 54: 908-932. The mature spermatozoan of Rossia pacifica consists of acrosome, nucleus, mitochondrial spur, and tail. Anteriorly, the complex acrosome has a cup-shaped depression containing membrane-bound vacuoles. The acrosome is enclosed by a meshwork of longitudinal and circumferential filaments; its invaginated posterior end surrounds juxtanuclear periacrosomal material. The elongated nucleus, of condensed chromatin filaments, encloses lateroposteriorly the proximal and distal centrioles withina centriolar fossa. Mitochondria lie in a separate, spurlike appendage; proximally its lateral margins are continuous with a collarlike annulus. Nine coarse outer fibres surround the 9 + 2 axoneme. They originate from a centriolar ring and diminish in diameter distally, Staining with periodic acid -Schiff demonstrates some glycogen in the mitochondrial spur near its junction with the nucleus.Structural significance and possible functional roles of the various elements are discussed relevant to fertilization. Although this sperm appears intermediate between the more primitive invertebrate sperm type and highly specialized vertebrate sperm, its morphology seems more related to fertilization biology than to phylogenetic position. The singular separateness of areas of energy production and use in R . pacifica sperm and the lengthy energy-transport path may provide unique opportunities for analysis of metabolic processes common to but indecipherable in other sperm. FIELDS, W. G., et K. A. THOMPSON. 1976. Ultrastructure and functional morphology of spermatozoa of Rossia pacifica (Cephalopoda, Decapoda). Can. J. Zool. 54: 908-932.Le spermatozoIde a maturiti, chez Rossia pacifica, contient I'acrosome, le noyau, l'eperon mitochondrial et la queue. Vers I'avant, I'acrosome complexe porte une depression en forme de coupe ou se trouvent les vacuoles reliees i la membrane. L'acrosome est retenu par un rtseau de filaments longitudinaux et circulaires; son extrtmite posterieure invaginee entoure la substance periacrosomienne juxtanuclCaire. Le noyau allonge, constitue de filaments compacts de chromatine, contient, en position posterieure laterale, les centrioles proximal et distal, ainsi qu'une fosse centriolaire. Les mitochondries occupent un appendice sipare, en forme d'eperon; dans la partie proximale de cet appendice, les bordures laterales sont en continuit6 avec un annulus formant une sorte de col. Neuf grosses fibres externes entourent les 9 + 2 axonkmes.Elles prennent leur origine dans un anneau centriolaire et elles s'amenuisent en direction distale. La coloration au I'acide periodique -Schiff revele la presence de glycogene dans l'eperon des mitonchondries, au voisinage de sa jonction avec le noyau.On discute de l'importance structurale et des r6les fonctionnels possibles de ces divers elements dans la fecondation. Bien que ce spermatozo~de apparaisse comme un intermediaire...

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Section: Discussionsupporting

confidence: 73%

“…Penetration is achieved by action of lysins and (or) extension of an acrosomal process. Some sperm have been found to contain several lysins (Heller and Raftery 1973). Vacuoles of the apical complex of R. pac$ca sperm may contain substances that aid in the dehiscence and (or) dissolution of membranes.…”

Section: Discussionmentioning

confidence: 99%

Ultrastructure and functional morphology of spermatozoa of Rossia pacifica (Cephalopoda, Decapoda)

Fields¹,

Thompson²

1976

Can. J. Zool.

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“…We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported Vitelline layer lysins are found in the sperm of a variety of animals such as Megathura [1,2], Tegula [3,4], Hydroides [5], Mytilus [6], Bufo [7] and some mammals [8 -lo]. Although it has long been throught that sea urchin sperm too have a vitelline layer lysin, only a brief report is available [ll].…”

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confidence: 99%

Purification and Characterization of a Chymotrypsin‐Like Enzyme from Sperm of the Sea Urchin, Hemicentrotus puleherrimus

Yamada

Matsui

Aketa

1982

European Journal of Biochemistry

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A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogenous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18 500 -19 000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N-benzoyl-L-arginine ethyl ester, N-a-toluenesulfonyl-L-arginine methyl ester, N-a-benzoyl-DL-argininep-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeS02F), chymostatin and L-I-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCHKl), and partially by soybean trypsin inhibitor and N-a-p-tosyl-L-lysine chloromethyl ketone (TosLysCHzCl). The enzyme is activated by CaC12, MgC12, NaCl and KCl, and loses its activity in 5 min at 67 "C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSOzF, TosPheCHzCl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2C1, and al-antitrypsin. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported Vitelline layer lysins are found in the sperm of a variety of animals such as Megathura [1,2], Tegula [3,4], Hydroides[5], Mytilus [6], Bufo [7] and some mammals [8 -lo]. Although it has long been throught that sea urchin sperm too have a vitelline layer lysin, only a brief report is available [ll]. A trypsin-like enzyme has been purified from sperm of Strongylocentrotuspurpuratus, but it was not shown whether this enzyme has the vitelline layer lytic activity [12]. According to a recent report [13], an arylsulfatase exists in both spermatozoa and seminal plasma of Hemicentrotus pulcherrimus and Strongylocentrotus intermedius and digests the jelly coat and vitelline layer. It is also suggested that a chymotrypsin-like enzyme participates in sperm penetration [14].In the preceding paper [15], we have shown that a chymotrypsin-like enzyme extracted from H. pulcherrimus sperm partially digests the vitelline layer, either intact or isolated, of the same species. Disruption by this enzyme preparation of some protein components of the vitelline layer has also been demonstrated by sodium dodecylsulfate/polyacrylamide gel electrophoresis. In this paper, purification and partial characterization of this enzyme is described. MATERIALS AND METHODS Crude ExtractGametes of the sea urchin Hemicentrotus pulcherrimus were...

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“…We examined whether the present factor has the vitelline layer lytic activity. The activity can be estimated by the turbidimetric method [Haino and Kigawa, 1969;Heller and Raftery, 1973;Yamada and Aketa, 19811 or by the observation of ultrastructural changes of the egg surface (eg, elongation of microvilli) [Yamada and Aketa, 1981;Yamada et al, 19821. Our factor did not decrease the turbidity of the vitelline layer suspension: The treatment of jellyless eggs with our factor caused no drastic changes such as elongation of microvilli.…”

Section: Discussionmentioning

confidence: 99%

A species‐specific sperm factor dispersing the jelly coat of the egg of the sea urchin, Anthocidaris crassispina

Yamada

Aketa

1983

Gamete Research

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A species-specific factor capable of dispersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassispina. It does not exert its effect on the vitelline layer. The purification has been accomplished by a four-step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL-4B, ion-exchange column chromatography on DEAE-cellulose, and affinity column chromatography on heparin-Sepharose CL-6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 0-rnercaptoethanol, estimated molecular weight being about 140,000.The jelly dispersion by the present factor is activated by CaCI2, and inhibited by KCI, MnCI2, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose-6-sulfate. Inorganic sulfates such as (NH&S04 and Na2S04 have no effect on jelly dispersion. This factor is heat-labile, losing its activity in 30 min at 50°C.The present factor is found also in the seminal plasma, and released from sperm themselves by treatment with Triton X-100. These results suggest that this factor is loosely bound to the sperm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly dispersing factor.Only trypsin and or-chymotrypsin among commercial enzymes tested disperse the jelly coat, but neither activity is detectable in our preparation. The jelly-dispersing activity is inhibited neither by trypsin inhibitors such as N-a-p-tosyl-L-lysine-chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L-I-tosylamide-2-phenyl-ethylchloromethyl ketone and chymostatin. Participation of trypsin-like and chymotrypsin-like enzymes in jelly dispersion seems unlikely.

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Isolation and purification of three egg-membrane lysins from sperm of marine invertebrate Megathura crenulata (giant keyhole limpet)

Cited by 17 publications

References 3 publications

Ultrastructure and functional morphology of spermatozoa of Rossia pacifica (Cephalopoda, Decapoda)

Ultrastructure and functional morphology of spermatozoa of Rossia pacifica (Cephalopoda, Decapoda)

Purification and Characterization of a Chymotrypsin‐Like Enzyme from Sperm of the Sea Urchin, Hemicentrotus puleherrimus

A species‐specific sperm factor dispersing the jelly coat of the egg of the sea urchin, Anthocidaris crassispina

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