The extracellular matrix of echinoderm and amphibian eggs: Visualization in quick-frozen, deep-etched, and rotary-shadowed specimens

1990 ◽  
Vol 48 (3) ◽  
pp. 60-61
Author(s):  
Barry Bonnell ◽  
Carolyn Larabell ◽  
Douglas Chandler
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Sea Urchin ◽  
Crystalline Structure ◽  
Cortical Granule ◽  
Two Dimensional ◽  
Small Peptides ◽  
Deep Etching ◽  
Quick Freezing ◽  
Acrosomal Reaction

Eggs of many species including those of echinoderms, amphibians and mammals exhibit an extensive extracellular matrix (ECM) that is important both in the reception of sperm and in providing a block to polyspermy after fertilization.In sea urchin eggs there are two distinctive coats, the vitelline layer which contains glycoprotein sperm receptors and the jelly layer that contains fucose sulfate glycoconjugates which trigger the acrosomal reaction and small peptides which act as chemoattractants for sperm. The vitelline layer (VL), as visualized by quick-freezing, deep-etching, and rotary-shadowing (QFDE-RS), is a fishnet-like structure, anchored to the plasma membrane by short posts. Orbiting above the VL are horizontal filaments which are thought to anchor the thicker jelly layer to the egg. Upon fertilization, the VL elevates and is transformed by cortical granule secretions into the fertilization envelope (FE). The rounded casts of microvilli in the VL are transformed into angular peaks and the envelope becomes coated inside and out with sheets of paracrystalline protein having a quasi-two dimensional crystalline structure.

Cortical Granule Exocytosis and Fertilization Coat Elevation is Reduced in Aging Sea Urchin Eggs

2000 ◽  
Vol 6 (S2) ◽  
pp. 966-967
Author(s):  
Amitabha Chakrabarti ◽  
Heide Schatten
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Sea Water ◽  
Secretory Granules ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis ◽  
Granule Secretion

Cortical granules are specialized Golgi-derived membrane-bound secretory granules that are located beneath the plasma membrane in unfertilized sea urchin eggs. Upon fertilization cortical granules discharge in a reaction induced by calcium and release their contents between the plasma membrane and a thin vitelline layer that lines the plasma membrane. Microvilli at the plasma membrane elongate incorporting cortical granule membranes during elongation. The vitelline layer elevates and becomes the egg's fertilization coat that hardens and serves as physical block to polyspermy. While we do not understand the precise mechanisms that participate in cortical granule discharge it is believed that actin plays a role in this process. Because actin and calcium metabolism is affected in aging cells we investigated if cortical granule secretion is affected in aging sea urchin eggs.Lytechinus pictus eggs were obtained by intracoelomic injection of 0.5M KCI to release the eggs into sea water at 23°C.

scholarly journals Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3573
Author(s):  
Nunzia Limatola ◽  
Jong Tai Chun ◽  
Sawsen Cherraben ◽  
Jean-Louis Schmitt ◽  
Jean-Marie Lehn ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Early Development ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Cortical Granule ◽  
Cortical Actin ◽  
Phenotypic Changes ◽  
Sea Urchin Egg ◽  
Cortical Granule Exocytosis

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg’s fertilization response.

Degradation of an extracellular matrix: sea urchin hatching enzyme removes cortical granule-derived proteins from the fertilization envelope

1993 ◽  
Vol 104 (3) ◽  
pp. 929-938 ◽  
Author(s):  
N.M. Mozingo ◽  
L.R. Hollar ◽  
D.E. Chandler
Keyword(s):  
Extracellular Matrix ◽  
Early Development ◽  
Sea Urchin ◽  
Ultrastructural Changes ◽  
Cortical Granule ◽  
Central Layer ◽  
Hatching Enzyme

The sea urchin fertilization envelope is an extracellular matrix assembled at fertilization to prevent polyspermy and protect the embryo during early development. During hatching, the embryo secretes a proteolytic hatching enzyme which dissolves the fertilization envelope, allowing a ciliated blastula to swim free. In this study we examined ultrastructural changes in the fertilization envelope during degradation of this matrix by hatching enzyme. The completed fertilization envelope is a trilaminar structure consisting of a dense, central layer of filaments sandwiched between surface coats of paracrystalline material. Hatching enzyme disassembles this matrix by degrading the paracrystalline layers and removing macromolecules from the central layer leaving behind a thin matrix of loosely woven fibers.

scholarly journals The vitelline layer of the sea urchin egg and its modification during fertilization. A freeze-fracture study using quick-freezing and deep-etching.

1980 ◽  
Vol 84 (3) ◽  
pp. 618-632 ◽  
Author(s):  
D E Chandler ◽  
J Heuser
Keyword(s):  
Sea Urchin ◽  
Freeze Fracture ◽  
Early Embryo ◽  
Coating Process ◽  
Deep Etching ◽  
Secondary Axis ◽  
Sea Urchin Egg ◽  
Quick Freezing ◽  
Coat Pattern ◽  
Fracture Study

Eggs of the sea urchin Strongylocentrotus purpuratus were quick-frozen, freeze fractured, and deep-etched to reveal the detailed structure of the vitelline layer (VL), an extracellular coat. The VL consisted of a network of fibers lying in sheet raised 20 nm off the plasma membrane and connected to it by a series of short processes. Sperm attached to the fibers of this sheet and upon fertilization the VL rose off the egg surface to form the fertilization envelope (FE). By 1 min postinsemination (p.i.), the FE had become augmented by a new set of smaller fibrils, and the original fibers of the VL appeared to be undergoing degradation. The FE exhibited casts of microvilli the VL had once covered. These were rounded at 1 min p.i., but by 2 min they had become angular and coated with an orderly array of repeating macromolecular units. In areas between casts, the coating process was slower; incomplete rows of units were seen at 5 min p.i. and complete rows at 10 min. Deep-etching of FE isolated from eggs by homogenization and differential centrifugation showed that both top and bottom surfaces were coated. The coat pattern was made up of 17.5-nm wide rows of parallelogram-like units that repeated every 12.2 nm along the row axis. Units in adjacent rows were in register to produce a secondary axis 76 degrees from the row axis. The results of this and previous studies suggest that the coating process plays a major role in "hardening" the FE to produce a tough barrier that protects the early embryo from chemical and mechanical injury.

Immune Deposits in the Glomerular Extracellular Matrix Detected by the Quick-Freezing and Deep-Etching Method

Nephron ◽  
1992 ◽  
Vol 62 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Koh Nakazawa ◽  
Shinichi Ohno ◽  
Atsuhiko Naramoto ◽  
Hiroya Takami ◽  
Hui-Jun Duan ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Deep Etching ◽  
Immune Deposits ◽  
Etching Method ◽  
Quick Freezing ◽  
Glomerular Extracellular Matrix

scholarly journals Evidence for involvement of metalloendoproteases in a step in sea urchin gamete fusion.

1988 ◽  
Vol 107 (2) ◽  
pp. 539-544 ◽  
Author(s):  
J L Roe ◽  
H A Farach ◽  
W J Strittmatter ◽  
W J Lennarz
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Sea Urchin ◽  
Acrosome Reaction ◽  
Fusion Process ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Gamete Fusion ◽  
Cortical Granule Exocytosis ◽  
Cytoplasmic Continuity

Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.

Cortical granule exocytosis is triggered by different thresholds of calcium during fertilisation in sea urchin eggs

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 55-63 ◽  
Author(s):  
John C. Matese ◽  
David R. McClay
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Calcium Release ◽  
Threshold Concentration ◽  
Calcium Wave ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis

SummaryIn sea urchin eggs, fertilisation is followed by a calcium wave, cortical granule exocytosis and fertilisation envelope elevation. Both the calcium wave and cortical granule exocytosis sweep across the egg in a wave initiated at the point of sperm entry. Using differential interference contrast (DIC) microscopy combined with laser scanning confocal microscopy, populations of cortical granules undergoing calcium-induced exocytosis were observed in living urchin eggs. Calcium imaging using the indicator Calcium Green-dextran was combined with an image subtraction technique for visual isolation of individual exocytotic events. Relative fluorescence levels of the calcium indicator during the fertilisation wave were compared with cortical fusion events. In localised regions of the egg, there is a 6s delay between the detection of calcium release and fusion of cortical granules. The rate of calcium accumulation was altered experimentally to ask whether this delay was necessary to achieve a threshold concentration of calcium to trigger fusion, or was a time-dependent activation of the cortical granule fusion apparatus after the ‘triggering’ event. Calcium release rate was attenuated by blocking inositol 1,4,5-triphospate (InsP3)-gated channels with heparin. Heparin extended the time necessary to achieve a minimum concentration of calcium at the sites of cortical granule exocytosis. The data are consistent with the conclusion that much of the delay observed normally is necessary to reach threshold concentration of calcium. Cortical granules then fuse with the plasma membrane. Further, once the minimum threshold calcium concentration is reached, cortical granule fusion with the plasma membrane occurs in a pattern suggesting that cortical granules are non-uniform in their calcium sensitivity threshold.

scholarly journals Changes in the topography of the sea urchin egg after fertilization.

1976 ◽  
Vol 71 (1) ◽  
pp. 35-48 ◽  
Author(s):  
E M Eddy ◽  
B M Shapiro
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Sea Urchin ◽  
Cytochalasin B ◽  
Cortical Granule ◽  
Sea Urchin Egg ◽  
Membrane Reorganization ◽  
Fertilization Membrane ◽  
Cortical Granule Exocytosis ◽  
Cytoskeletal Elements

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.

Ultrastructural study of cytoskeletal interactions with endosomes in macrophages by quickfreezing and deep-etching method

1990 ◽  
Vol 48 (3) ◽  
pp. 576-577
Author(s):  
Takeshi Baba ◽  
Nobuki Shiozawa ◽  
Masao Hotch ◽  
Shinichi Ohno
Keyword(s):  
Immune Complex ◽  
Ultrastructural Study ◽  
Fc Receptor ◽  
Coated Pits ◽  
Deep Etching ◽  
Receptor Mediated Endocytosis ◽  
Etching Method ◽  
Quick Freezing

Endosomes are vesicular or tubular organelles that play important roles in transports of receptors and receptor―bound ligands during receptor-mediated endocytosis. The mechanisms of endocytic transports from clathrin-coated pits to lysosomes have been studied by many investigators. However, few studies were reported about the interactions between endosomes and cytoskeletons. In this study, Fc-receptor-mediated endocytosis in macrophages are investigated by quick-freezing and deep-etching (QF-DE) method combined with gold-labeled immune complex and “replica scraping method”.

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