Isolation and characterization of the vitelline layer of sea urchin eggs.

Glabe, Charles G.; Vacquier, Victor D.

doi:10.1083/jcb.75.2.410

Cited by 83 publications

(36 citation statements)

References 36 publications

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“…The same method as described in the preceding paper [15] was used. The vitelline layers isolated after the method of Glabe and Vaquier [21] were sonicated for 1 min in an ice bath and the change in turbidity induced by our preparation was estimated at 640 nm with a Hitachi 624 spectrophotometer at room temperature (23 -…”

Section: Detection Of the Vitelline Layer Lytic Activitymentioning

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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Section: Detection Of the Vitelline Layer Lytic Activitymentioning

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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“…polyacrylamide gel electrophoresis of vitelline coats prepared from the injected ovaries showed fucose incorporation into the same three glycoproteins present in vitelline coats from mature oocytes and identified by their affinity for '5I-labeled fucosebinding protein [ Experimental evidence obtained in several animals (1)(2)(3)(4)(5)(6)(7) indicates that the vitelline coat (VC) (and its equivalent zona pellucida in the mammalian egg) bears the primary sperm receptors-i. e., the molecular complexes that enable specific recognition and binding of spermatozoa to the egg.…”

mentioning

confidence: 99%

Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciona intestinalis (Ascidia).

Rosati¹,

Cotelli²,

Santis³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The sperm receptors of the ascidian oocyte are located at the outer surface of the vitelline coat (formerly called the chorion). The fucose residues are the receptor's most important components for sperm recognition and binding. We asked whether the fucosyl-containing glycoproteins of the vitelline coat are a product ofthe oocyte, the follicle cells, or the test cells. Ovaries of Ciona intestinalis were injected with L-[3H]fucose and the progress of its incorporation was followed by using autoradiography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the injected gonads and of the isolated vitelline coats. We found that incorporation of fucose begins within the vitellogenic oocytes, and fucose slowly accumulates in the differentiating vitelline coat. At no time could fucose incorporation be detected Experimental evidence obtained in several animals (1-7) indicates that the vitelline coat (VC) (and its equivalent zona pellucida in the mammalian egg) bears the primary sperm receptors-i. e., the molecular complexes that enable specific recognition and binding of spermatozoa to the egg. The VC is usually considered to be a product of the follicle cells. Should this be the case, it implies that the synthesis of the sperm receptors, which play a key role in the process of sperm-egg interaction, is delegated to the somatic components ofthe gonad.In mammals the primary sperm receptors are a component of the zona pellucida, which makes it analogous to the VC of, for example, the sea urchin egg. Recently, Bleil and Wassarman (8) have provided convincing evidence that the zona pellucida is synthesized by the oocyte.The ascidian egg is convenient for studying functional relationships between the oocyte and its envelopes. The latter consist ofa fibrous chorion and a layer oflarge follicle cells attached to its outer surface. Between the chorion and the surface of the oocyte there is a perivitelline space filled with the test cells, whose significance is still unclear. The chorion can be manually separated (with fine needles) from the egg. This offers an almost unique opportunity to study "naked" eggs and egg envelopes without the use of enzyme treatments. Work from this laboratory has shown that the chorion is the carrier of the primary sperm receptors (9, 10); hence, we have suggested that it be designated as the VC of the egg (11). There are no other membranes or coats at the surface of the egg (12).In a previous study (13) we described the differentiation of the VC ofthe oocyte of Ciona intestinalis during oogenesis. We demonstrated that the precursor material of the VC (VCPM) of Ciona oocytes first appears soon after the oocyte and its accompanying cells have left the germinal strand and is in the form offluffy fibrillar patches at the surface of the oocyte. When the oocyte enters the vitellogenic stage, the patches merge with one another, thus giving rise to a coat ofloosely arranged fibrils that surrounds the oocyte. At this stage a continuous layer, identifiable as the VC, becomes visible ev...

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“…We sometimes observed immunoreactive proteins of lower molecular weight at 220, 190, 180, 140, and 105 kDa in the sample eluted from the immunoaffinity column. It is well known that sea urchin eggs contain a trypsin-like protease activity (32). However, in spite of the addition of protease inhibitors, we could not totally block degradation of the receptor protein.…”

Section: Identification Of Sulfated Neu5gc and Neu5gcmentioning

confidence: 71%

Identification of sulfated oligosialic acid units in theO-linked glycan of the sea urchin egg receptor for sperm

Kitazume-Kawaguchi

Inoue

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The Strongylocentrotus purpuratus sea urchin egg receptor for sperm is a cell surface glycoprotein with a molecular mass of 350 kDa. Recent studies indicate that the sulfated O-linked glycans isolated from the receptor bind to acrosome-reacted sperm. The purified receptor was analyzed with respect to amino acid and carbohydrate content and shown to be composed of 70% carbohydrate by weight. Compositional analysis indicated that both N-and O-linked oligosaccharide chains were present. After peptide:N-glycanase treatment of the receptor to remove most of the N-linked glycan chains, the majority of the sialic acid residues remained associated with the receptor and were shown by several types of experiments to be composed of sulfated oligosialic acid units attached to the O-linked glycan chains of the receptor. Chemical and physical studies on oligosialic chains discovered earlier in the Pronase-generated glycopeptide fraction isolated from the egg cell surface complex of another species of sea urchin, Hemicentrotus pulcherrimus, established that these molecules had the structure: (SO 4 ؊ )-9Neu5Gc␣2(35-O glycolyl Neu5Gc␣23) n . Based on comparative and analytical studies, it was concluded that this sulfated oligosaccharide is a component of a GalNAc-containing chain that is O-linked to the polypeptide chain of the sea urchin egg receptor for sperm. Using a competitive inhibition of fertilization bioassay it was shown that the sulfated oligosialic acid chains derived from the S. purpuratus egg cell surface complex inhibited fertilization; the nonsulfated form of this oligosialic chain had little inhibitory activity.In fertilization, cell surface molecules of the egg and sperm play a central role in the species-specific interactions that occur in many organisms (1, 2). In the case of the sea urchin, earlier studies showed that the sperm protein, bindin, which is a component of the acrosome granule, plays a key role in gamete recognition (3, 4). Following early work in which a high molecular weight glycoprotein on the egg cell surface was implicated as a receptor for sperm (5-7), it was shown that Pronase-generated glycopeptides prepared from this crude receptor preparation inhibited fertilization, but without species specificity (8, 9). Later, a cell surface glycoprotein with a molecular mass of 350 kDa was identified as the egg receptor in Strongylocentrotus purpuratus (10,11). This molecule, as well as a 70-kDa extracellular fragment generated by lysyl endoproteinase C digestion of intact eggs, were shown to contain sugars typical of both N-and O-linked oligosaccharides, as well as sulfate (10, 12). These oligosaccharide chains were fractionated, and the putative O-linked chains were shown to inhibit fertilization and bind without species specificity to acrosome-reacted, but not to unreacted sperm (13). In a subsequent study it was shown that attachment of these chains via a neoglycoprotein to beads mediated kinetically stable binding of sperm to such beads (14).In earlier studies, ␣235-O glycolyl -linked p...

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Isolation and characterization of the vitelline layer of sea urchin eggs.

Cited by 83 publications

References 36 publications

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

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

Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciona intestinalis (Ascidia).

Identification of sulfated oligosialic acid units in theO-linked glycan of the sea urchin egg receptor for sperm

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