Bindin is essential for fertilization in the sea urchin

Species-specific sperm−egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms is under particularly stringent conditions, since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome results in insoluble particles that cause homospecific eggs to aggregate, whereas no aggregation occurs with heterospecific eggs. Therefore, Bindin is concluded to play a critical role in fertilization, yet its function has never been tested. Here we report that Cas9-mediated inactivation of the bindin gene in a sea urchin results in perfectly normal-looking embryos, larvae, adults, and gametes in both males and females. What differed between the genotypes was that the bindin−/− sperm never fertilized an egg, functionally validating Bindin as an essential gamete interaction protein at the level of sperm–egg cell surface binding.

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Inhibition of Cell Surface Binding of Fibronectin and Fibronectin-Promoted Cell Migration by Synthetic Peptides in Sea Urchin Primary Mesenchyme Cells In Vitro. (cell migration/fibronectin/synthetic peptides/primary mesenchyme cells)

Development Growth & Differentiation ◽

10.1111/j.1440-169x.1987.00579.x ◽

1987 ◽

Vol 29 (6) ◽

pp. 579-589 ◽

Cited By ~ 12

Author(s):

HIDEKI KATOW

Keyword(s):

Cell Migration ◽

Cell Surface ◽

Sea Urchin ◽

Synthetic Peptides ◽

Surface Binding ◽

Cell Surface Binding ◽

Mesenchyme Cells ◽

Primary Mesenchyme Cells

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The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor

The Journal of Cell Biology ◽

10.1083/jcb.122.4.887 ◽

1993 ◽

Vol 122 (4) ◽

pp. 887-895 ◽

Cited By ~ 41

Author(s):

K Ohlendieck ◽

ST Dhume ◽

JS Partin ◽

WJ Lennarz

Keyword(s):

Cell Surface ◽

Sea Urchin ◽

Transmembrane Protein ◽

Biologically Active ◽

Cell Surface Receptor ◽

Egg Cell ◽

Dependent Manner ◽

Binding Studies ◽

Isolation And Characterization ◽

Species Specific

The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.

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

The Journal of Cell Biology ◽

10.1083/jcb.116.3.647 ◽

1992 ◽

Vol 116 (3) ◽

pp. 647-658 ◽

Cited By ~ 39

Author(s):

K R Foltz ◽

W J Lennarz

Keyword(s):

Cell Surface ◽

Sea Urchin ◽

Egg Cell ◽

Cortical Granule ◽

Dependent Manner ◽

Western Blots ◽

Cell Surface Glycoprotein ◽

Sperm Binding ◽

Sea Urchin Egg ◽

Species Specific

Sea urchin egg fertilization requires the species-specific interaction of molecules on the sperm and egg surfaces. Previously, we isolated an extracellular, 70-kD glycosylated fragment of the S. purpuratus egg receptor for sperm by treating the eggs with lysylendoproteinase C (Foltz, K. R., and W. J. Lennarz. 1990. J. Cell Biol. 111:2951-2959). To characterize the receptor further, we have generated a polyclonal antiserum (anti-70KL) against the purified 70-kD fragment. Anti-70KL was found to react with a single polypeptide of approximately 350 kD on Western blots, presumed to be the intact receptor, in an egg cell surface preparation. This polypeptide appeared to be tightly associated with the plasma membrane/vitelline layer complex, as it was released from these preparations only by detergent treatment. Immunofluorescence microscopy revealed that the receptor was distributed evenly over the egg surface. The anti-70KL was species specific both in its ability to recognize the egg surface protein and to inhibit sperm binding. Fab fragments generated from affinity-purified anti-70KL also bound to the egg surface and inhibited sperm binding in a concentration-dependent manner. Interestingly, treatment with Fabs caused a small percentage of eggs to undergo cortical granule exocytosis, even in the absence of external Ca2+. These results confirm earlier findings indicating that the receptor is a cell surface glycoprotein of high molecular weight that species specifically binds sperm. This antiserum provides a powerful tool for further investigation of gamete interactions and the structure of the sperm receptor.

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Immunogold and immunoblot studies on a cell surface protein found in primary mesenchyme cells and in the cortical secretory vesicles of the sea urchin egg

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128444 ◽

1987 ◽

Vol 45 ◽

pp. 832-833

Author(s):

G.L. Decker ◽

M.C. Valdizan

Keyword(s):

Cell Surface ◽

Sea Urchin ◽

Surface Protein ◽

Egg Cell ◽

Secretory Vesicles ◽

Cell Surface Protein ◽

Surface Complexes ◽

Mesenchyme Cells ◽

Lowicryl K4m ◽

Primary Mesenchyme Cells

A monoclonal antibody designated MAb 1223 has been used to show that primary mesenchyme cells of the sea urchin embryo express a 130-kDa cell surface protein that may be directly involved in Ca2+ uptake required for growth of skeletal spicules. Other studies from this laboratory have shown that the 1223 antigen, although in relatively low abundance, is also expressed on the cell surfaces of unfertilized eggs and on the majority of blastomeres formed prior to differentiation of the primary mesenchyme cells.We have studied the distribution of 1223 antigen in S. purpuratus eggs and embryos and in isolated egg cell surface complexes that contain the cortical secretory vesicles. Specimens were fixed in 1.0% paraformaldehyde and 1.0% glutaraldehyde and embedded in Lowicryl K4M as previously reported. Colloidal gold (8nm diameter) was prepared by the method of Mulpfordt.

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