Heat Sensitivity of the Cortical Granule Protease From Hamster Eggs

Gwatkin, Ralph B. L.; Williams, D. T.

doi:10.1530/jrf.0.0390153

Cited by 20 publications

(12 citation statements)

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“…Studies with golden hamster gametes in vitro have shown that incubation of eggs or isolated zonae pellucidae in cortical granule material will prevent spermatozoa from binding to them (Barros & Yanagimachi, 1971. The active agent in the cortical granule exúdate is heatlabile (Gwatkin & Williams, 1974) and appears to be a trypsin-like protease, since its action is blocked by specific trypsin inhibitors (Gwatkin, Williams, Hartmann & Kniazuk, 1973). Other experiments have shown that the receptor for spermatozoa in the zona pellucida is sensitive to trypsin and chymotrypsin (Hartmann & Gwatkin, 1971), but not to glycosidases or upases (Gwatkin, Williams & Andersen, 1973).…”

Section: Introductionmentioning

confidence: 99%

Induction of the cortical reaction in hamster eggs by membrane-active agents

Gwatkin¹,

Rasmusson²,

Williams³

1976

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Summary. Fusion of capacitated spermatozoa with the vitelline membrane, but not actual penetration, appears to initiate the cortical reaction in hamster eggs. The reaction can be artificially induced by the application of positively charged particles to the vitelline surface, a situation which may normally be prevented by the zona pellucida. Exposure of hamster eggs to neuraminidase, to lectins (concanavalin A and phytohaemagglutinin-P), to a monovalent ionophore (boromycin) and to 1,3-bis(4-chlorocinnamylideneamino)guanidine elicits a cortical granule discharge resulting in a block to fertilization. These agents all appear to act by inducing depolarization of the vitelline membrane.

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Section: Introductionmentioning

confidence: 99%

Induction of the cortical reaction in hamster eggs by membrane-active agents

Gwatkin¹,

Rasmusson²,

Williams³

1976

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“…Sea urchin (20) and mammalian (21) eggs release trypsin-like protease activity shortly after fusion with sperm. The protease is believed to aid in prevention of polyspermic fertilization by digesting hypothetical sperm receptors, thus rendering the egg surface incapable of sperm adhesion (11,12,21). Unfertilized eggs treated with fertilization protease rapidly lose their ability to bind sperm and to be fertilized (11,12,21), although they can still be activated by A23187 (11).…”

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confidence: 99%

“…The protease is believed to aid in prevention of polyspermic fertilization by digesting hypothetical sperm receptors, thus rendering the egg surface incapable of sperm adhesion (11,12,21). Unfertilized eggs treated with fertilization protease rapidly lose their ability to bind sperm and to be fertilized (11,12,21), although they can still be activated by A23187 (11). The protease is inhibited by natural and synthetic inhibitors of pancreatic trypsin (11,12,20,21).…”

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confidence: 99%

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Egg surface glycoprotein receptor for sea urchin sperm bindin.

Glabe¹,

Vacquier²

1978

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

130

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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...

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“…By contrast, electrical stimulation of hamster eggs with a square-wave pulse of 150 V for 1 msec was said to be highly effective in eliciting the reaction (Gwatkin et al, 1973;Gwatkin & Williams, 1974). …”

Section: Breakdown Of Cortical Granulesmentioning

confidence: 98%