Fusion of membranes during fertilization. Increases of the sea urchin egg's membrane capacitance and membrane conductance at the site of contact with the sperm.

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.

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THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

The Journal of Cell Biology ◽

10.1083/jcb.3.1.103 ◽

1957 ◽

Vol 3 (1) ◽

pp. 103-110 ◽

Cited By ~ 20

Author(s):

Lord Rothschild

Keyword(s):

Plasma Membrane ◽

Sea Urchin ◽

Sea Water ◽

Unit Area ◽

Membrane Capacitance ◽

Percentage Reduction ◽

Cortical Granules ◽

Sea Urchin Egg

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.

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A localized zone of increased conductance progresses over the surface of the sea urchin egg during fertilization.

The Journal of General Physiology ◽

10.1085/jgp.97.3.579 ◽

1991 ◽

Vol 97 (3) ◽

pp. 579-604 ◽

Cited By ~ 11

Author(s):

D H McCulloh ◽

E L Chambers

Keyword(s):

Patch Clamp ◽

Sea Urchin ◽

Membrane Conductance ◽

Phase 2 ◽

Opposite Pole ◽

Sea Urchin Egg ◽

Conductance Changes ◽

Conductance Increase ◽

Membrane Patch ◽

Whole Egg

Although activation of a sea urchin egg by sperm leads to three phases of membrane conductance increase in the egg, the mechanism by which the sperm causes these conductance changes is not known. We used the loose patch clamp technique to localize the conductance changes in voltage clamped eggs. A patch of the egg's membrane was isolated from the bath by pressing the loose patch clamp pipette against the egg surface. Sperm added to the bath attached to the surface of the egg in a region other than at the isolated membrane patch. During phase 1 of the activation current, no changes of the membrane conductance were detected. At the time of, and subsequent to the onset of phase 2, large currents recorded between the interior of the patch pipette and the bath were attributed to changes of the seal resistance between the surface of the egg and the pipette. A local change of membrane conductance was observed during phase 2 despite the changes of seal resistance. During phase 2, the large amplitude and short duration of the local membrane conductance increase relative to the membrane, conductance increase for the whole egg during phase 2 indicated that the conductance increase occurred over the entire surface of the egg, but not simultaneously. The time when the peak conductance for the membrane patch occurred, relative to the time of onset for phase 2 in the whole egg, depended on the distance, measured in a straight line, between the site of sperm attachment and the tip of the pipette. These data indicate that the localized conductance increase progressed over the surface of the egg from the site of sperm attachment to the opposite pole of the egg. It is proposed that the local conductance increase, the cortical reaction, and the change of seal resistance are all evoked by a common cytoplasmic message that progresses throughout the cytoplasm of the egg from the site of sperm attachment to the opposite pole of the egg.

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Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

The Journal of Cell Biology ◽

10.1083/jcb.134.2.329 ◽

1996 ◽

Vol 134 (2) ◽

pp. 329-338 ◽

Cited By ~ 35

Author(s):

S S Vogel ◽

P S Blank ◽

J Zimmerberg

Keyword(s):

Poisson Process ◽

Sea Urchin ◽

Physiological Responses ◽

Cortical Granule ◽

Cortical Granules ◽

Granule Exocytosis ◽

Calcium Dependence ◽

Sea Urchin Egg ◽

High Calcium ◽

Cortical Granule Exocytosis

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

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Isolation and partial characterization of the plasma membrane of the sea urchin egg.

The Journal of Cell Biology ◽

10.1083/jcb.87.1.248 ◽

1980 ◽

Vol 87 (1) ◽

pp. 248-254 ◽

Cited By ~ 54

Author(s):

W H Kinsey ◽

G L Decker ◽

W J Lennarz

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Sea Urchin ◽

Plasma Membranes ◽

Binding Property ◽

Marker Enzyme ◽

Surface Complex ◽

Sea Urchin Egg ◽

Peripheral Proteins ◽

Cortical Vesicles

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.

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Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation

Developmental Biology ◽

10.1016/0012-1606(72)90070-x ◽

1972 ◽

Vol 29 (3) ◽

pp. 307-320 ◽

Cited By ~ 33

Author(s):

H. Schuel ◽

W.L. Wilson ◽

R.S. Bressler ◽

J.W. Kelly ◽

J.R. Wilson

Keyword(s):

Sea Urchin ◽

Cortical Granules ◽

Sea Urchin Egg

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DIRECT ISOLATION OF THE HYALINE LAYER PROTEIN RELEASED FROM THE CORTICAL GRANULES OF THE SEA URCHIN EGG AT FERTILIZATION

The Journal of Cell Biology ◽

10.1083/jcb.45.3.615 ◽

1970 ◽

Vol 45 (3) ◽

pp. 615-622 ◽

Cited By ~ 49

Author(s):

R. E. Kane

Keyword(s):

Protein Content ◽

Sea Urchin ◽

Insoluble Fraction ◽

Glycerol Solution ◽

Cortical Granules ◽

Simple Method ◽

Quantitative Measurements ◽

Hawaiian Species ◽

Sea Urchin Egg

Treatment of the eggs of the sea urchin with a 1 M solution of glycerol at fertilization allows the recovery from this solution of the protein released from the cortical granules, including that which would normally give rise to the hyaline layer. The calcium-gelable protein previously extracted from whole eggs and from isolated cortical material was found to be present in the glycerol solution, confirming its localization in the cortical granules and its role in the hyaline layer. Quantitative measurements on the eggs of two Hawaiian species, Colobocentrotus atratus and Pseudoboletia indiana, which have the widest variation in the gel protein content, demonstrated that a proportionate amount of this material was released at fertilization in these species, which correlates with the thickness of the hyaline layer in the two cases. In addition, the calcium-insoluble fraction of Sakai can be extracted from these eggs after removal of the hyaline protein by glycerol, showing that this is a different material. A simple method for the separation of the hyaline protein from the calcium-insoluble fraction in solution is provided.

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A COMPARATIVE STUDY OF THE ISOLATION OF THE CORTEX AND THE ROLE OF THE CALCIUM-INSOLUBLE PROTEIN IN SEVERAL SPECIES OF SEA URCHIN EGG

The Journal of Cell Biology ◽

10.1083/jcb.41.1.133 ◽

1969 ◽

Vol 41 (1) ◽

pp. 133-144 ◽

Cited By ~ 32

Author(s):

R. E. Kane ◽

R. E. Stephens

Keyword(s):

Comparative Study ◽

Sea Urchin ◽

Protein Composition ◽

Cortical Region ◽

Cortical Granules ◽

Divalent Ions ◽

Sea Urchin Egg ◽

Protein Gelation ◽

Isolated Cortex

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.

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Isolated Plasma Membranes of Sea Urchin Eggs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066723 ◽

1971 ◽

Vol 29 ◽

pp. 534-535

Author(s):

S. Inoue ◽

E. C. Preddie ◽

P. Guerrier

Keyword(s):

Electron Microscope ◽

Sea Urchin ◽

Plasma Membranes ◽

Membrane System ◽

Vitelline Membrane ◽

Thin Sections ◽

Sea Urchin Eggs ◽

Sea Urchin Egg ◽

Discontinuous Sucrose Gradient ◽

Fertilization Membrane

From electron microscope studies of thin sections the sea urchin egg is known to be surrounded by the peripheral membrane system which is made up of the outer coat (vitelline membrane), which elevates from an egg surface after fertilization and becomes a part of the fertilization membrane, and the plasma membrane. In these experiments an effort has been made to isolate plasma membranes of sea urchin eggs and these isolated membranes were observed in the electron microscope.The vitelline membrane of the eggs from the sea urchin Strongylocentrotus purpuratus was at first digested away by the treatment with 0.02% trypsin in 0.01 M Tris-HCl buffer (pH 8.0) for 5 minutes at 28°C. The plasma membranes were then isolated according to the method of Song et al. which was used for the isolation of rat liver plasma membranes. The vitelline membrane-free eggs were gently homogenized in 10-3 M NaHC03 (pH 7.5) and freed membranes were collected by centrifugation over a discontinuous sucrose gradient preparation.

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