Egg surface glycoprotein receptor for sea urchin sperm bindin.

Abstract

Bindin is an insoluble protein coating the sperm acrosome process and mediating the adhesion of sperm to sea urchin eggs. Milligrams of bindin have been isolated. Here we report the identification, isolation, and partial characterization of a high molecular weight, trypsin-sensitive glycoprotein fraction from the sea urchin egg surface having species-specific affinity for bindin. This glycoprotein may be the egg surface receptor for bindin. The bindin receptor was released from 125I-labeled eggs by parthenogenetic activation of eggs with ionophore A23187 in the presence of soybean trypsin inhibitor. The receptor has an isoelectric point of 4.02 and a molecular weight in sea water >5 X 106, suggesting that it is an aggregate. It contains 34% neutral sugars, which are galactose and mannose.Attachment of spermatozoa to the egg surface occurs in many animal species, including mammals (1). In sea urchins, gamete adhesion is between the sperm acrosome process and the egg vitelline layer (2). An insoluble protein, named bindin, has been isolated from acrosome granules of sea urchin sperm. Peroxidase-conjugated antibody to bindin localizes bindin on the acrosome process'membrane and on the vitelline layer adjacent to the point of adhesion of the acrosome process (3). Bindin is a species-specific agglutinin of unfertilized eggs (4, 5). Trypsin-generated glycopeptides from egg surfaces block the eggagglutinating property of bindin and periodate oxidation of eggs renders them nonagglutinable by bindin. These data are evidence that bindin mediates sperm adhesion to eggs.The presence of species-specific glycoprotein "sperm receptors" on sea urchin eggs has been inferred for several years (6-12). Here we present the direct demonstration and isolation of a large, protease-sensitive egg surface glycoprotein having specific affinity for bindin. MATERIALS AND METHODSGametes and Bindin Isolation. Gametes of Strongylocentrotus purpuratus were obtained by pouring 0.5 M KCI into opened body cavities. Egg jelly coats were dissolved by 2-min exposure to pH 4.7 sea water, followed by settling and resuspension in pH 8.0 sea water. The pH 4.7 treatment was repeated and the washed eggs were stored at 4°. Bindin was isolated as described (4). After the final sea water wash the insoluble bindin pellet was resuspended in sea water by mild sonication, the protein concentration was determined (13), and 1-ml portions were stored at -700.125I Labeling of Egg Surfaces. Twenty milliliters of 50% (vol/vol) suspension of dejellied eggs in sea water containing 10 mM Tris (pH 8.0) was placed in a 50-ml conical tube. Two-tenths milliliter of 125I (carrier-free, 2.0 mCi/ml in 10 mM NaOH, Amersham) was added, followed by 0.1 ml of chloramine-T (10 mg/ml in sea water) at 15-sec intervals for 5 min. The reaction was stopped by adding 2.0 ml of sodium metabisulfite (10 mg/ml in sea water). The labeled eggs were washed a minimum of five times by settling through fresh sea water. 52I-Labeled eggs developed to the gastrula stage.Isolation of 125I-Label...

Secretory functions of egg cortical granules in fertilization and development: A critical review

“…Is it possibly an alteration in the electrical properties of the oolemma similar to that involved in the rapid block to polyspermy [Gould-Somero and Jaffe, 1977;Jaffe, 1976]? 19721 or with the purified cortical granule protease [Carroll andEpel, 1975a, 1975b;Vacquier et al, 19731 prevents sperm binding to the vitelline layer and blocks fertilization. These results taken together with other experimental evidence [Glabe and Vacquier, 1978;Shapiro, 1975;Schmell et al, 19771 suggest that one secretory function of the cortical granule protease during fertilization in echinoid eggs is to remove sperm receptor molecules from the vitelline layer. This process, which must take place coincidentally with the detachment of the vitelline layer from the oolemma [Longo and Schuel, 1973;Longo et al, 1974;Schuel et al, 1976aSchuel et al, , 1976b, is thought to be responsible for the blocking of polyspermy by 1) preventing the attachment of additional sperm to the egg, and 2) detaching previously bound sperm from the egg surface [Epel, 1975[Epel, , 1978Vacquier et al, 19731, since sperm binding to the egg is a prerequisite for gamete fusion and sperm entrance [Summers et al, 19751.…”

“…For somatic secretory cells the stimulus to degranulate is usually provided by the interaction of a humoral secretagogue with a specific receptor molecule in the cell's plasma membrane. The equivalent function is subserved during fertilization by the interaction of complementary macromolecules on the reacting gametes at the site of fusion [Glabe and Vacquier, 1978;Lillie, 1919;Metz, 1978;Schmell et al, 1977;Summers et al, 197.51. Both eggs and somatic cells exhibit similar responses to stimulation, namely depolarization of the plasma membrane and calciumtriggered release of secretory product by exocytosis (see sectiqns 4.1,4.2.2, and 5.2, above).…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…Upon egg activation, CGs fuse with the oocyte plasma membrane and release their contents into the perivitelline space; the CG enzymes then biochemically and structurally modify the VM so that sperm can no longer bind or remain bound to the egg. For instance, sea urchin CGs contain a serine protease that cleaves the sperm receptor, bindin, from the VM (Carroll and Epel, 1975;Glabe and Vacquier, 1978;Haley and Wessel, 1999). In mouse and sea urchin oocytes, CGs contain ovoperoxidases that harden the extracellular matrix by cross-linking tyrosine residues on adjacent proteins (Gulyas, 1979;Schmell and Gulyas, 1980;LaFleur et al, 1998).…”

Identification of the sea urchin egg receptor for sperm using an antiserum raised against a fragment of its extracellular domain.

“…Several groups have reported the isolation of high molecular weight glycoconjugates from the egg surface that inhibit sperm-binding activity, and therefore have been candidates for the receptor for sperm (Metz, 1978;Schmell et al ., 1977;Tsuzuki et al ., 1977;Glabe and Vacquier, 1978;Rossignol et al ., 1981Rossignol et al ., , 1984Yoshida and Aketa, 1983;AcevedoDuncan and Carroll, 1986) . However, none of these molecules has been further characterized .…”

“…It has been known for some time that the receptor for bindin is associated with the vitelline layer of the sea urchin egg (Summers and Hylander, 1975 ;Glabe and Vacquier, 1977b;Tsuzuki et al ., 1977). The simplest hypothesis has been one that equates the bindin receptor with the actual sperm receptor ; i .e., the molecule on the egg surface that species specifically recognizes acrosome-reacted sperm (Glabe and Vacquier, 1978) .…”