scholarly journals Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs.

1984 ◽  
Vol 99 (5) ◽  
pp. 1647-1654 ◽  
Author(s):  
A Eisen ◽  
D P Kiehart ◽  
S J Wieland ◽  
G T Reynolds
Keyword(s):  
Spatial Distribution ◽  
Sea Urchin ◽  
Pyridine Nucleotide ◽  
Membrane Depolarization ◽  
Temporal Sequence ◽  
Cortical Granule ◽  
Free Calcium ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis ◽  
Cytoplasmic Free Calcium

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at approximately 23 s as measured by calcium-aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at approximately 51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.

scholarly journals Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

1991 ◽  
Vol 113 (4) ◽  
pp. 769-778 ◽  
Author(s):  
T Whalley ◽  
I Crossley ◽  
M Whitaker
Keyword(s):  
Sea Urchin ◽  
Okadaic Acid ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Transduction Mechanisms ◽  
Egg Signal ◽  
Cortical Granule Exocytosis ◽  
Gamma S

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.

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.

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

2003 ◽  
Vol 301 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Juana López-Godı́nez ◽  
Teresa I. Garambullo ◽  
Guadalupe Martı́nez-Cadena ◽  
Jesús Garcı́a-Soto
Keyword(s):  
Sea Urchin ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis

The relation between ionized calcium and cortical granule exocytosis in eggs of the sea urchin Echinus esculentus

1980 ◽  
Vol 207 (1167) ◽  
pp. 149-161 ◽  
Keyword(s):  
Molecular Mass ◽  
Electric Fields ◽  
Sea Urchin ◽  
Cortical Granule ◽  
Free Calcium ◽  
Relative Molecular Mass ◽  
Cortical Granules ◽  
Full Complement ◽  
Free Calcium Concentration ◽  
Cortical Granule Exocytosis

By subjecting sea urchin eggs to intense, short-duration, electric fields the permeability to low relative molecular mass substances is markedly increased. After such treatment, the extracellular space markers 22 Na + and [ 14 C]mannitol penetrate into the interior of the egg and localized destruction of the oolemma is apparent. The technique permits the rapid introduction of low relative molecular mass substances into the interior of the egg. We have employed it to investigate the efficacy of various buffered calcium concentrations in bringing about exocytosis of cortical granules of the egg. Eggs rendered permeable in the presence of EGTA (free Ca < 10 -8 M) retain a full complement of cortical granules and appear little different in cortical ultrastructure from unfertilized eggs, as judged by scanning and transmission electron microscopy. The proportion of cortical granules remaining in the egg cortex 30 s after application of an electric field in the presence of higher concentrations of calcium decreases pro­gressively as the free calcium concentration introduced into the egg interior is increased from 0.5 to 6 μM. The disappearance of the cortical granules is attributed to their having undergone exocytosis, since the changes in cortical ultrastructure that result from treatment with micro­-molar calcium concentrations are demonstrated to be similar to the changes that result from exocytosis of the cortical granules in intact eggs after fertilization.

scholarly journals Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs.

1995 ◽  
Vol 131 (5) ◽  
pp. 1183-1192 ◽  
Author(s):  
T Whalley ◽  
M Terasaki ◽  
M S Cho ◽  
S S Vogel
Keyword(s):  
Sea Urchin ◽  
Fluorescent Dyes ◽  
Secretory Granules ◽  
Wave Length ◽  
Fluid Phase ◽  
Cortical Granule ◽  
Lipid Phase ◽  
Granule Exocytosis ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.

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

1996 ◽  
Vol 134 (2) ◽  
pp. 329-338 ◽  
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.

Dynamics of cortical granule exocytosis at fertilization in living mouse eggs

1996 ◽  
Vol 270 (5) ◽  
pp. C1354-C1361 ◽  
Author(s):  
M. Tahara ◽  
K. Tasaka ◽  
N. Masumoto ◽  
A. Mammoto ◽  
Y. Ikebuchi ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Granule ◽  
Free Calcium ◽  
Cortical Granules ◽  
Acetoxymethyl Ester ◽  
Living Mouse ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs

Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently we used an impermeable fluorescent membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), to develop a method to evaluate the kinetics of exocytosis in single living cells. In this study we used digital imaging and confocal laser scanning microscopy to evaluate CG exocytosis in living mouse eggs with TMA-DPH. Time-related changes of CG exocytosis were estimated as the percent increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate, and ceased at 45-60 min. Whereas the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next we used this method to determine some intracellular mediators of exocytosis in the egg. An intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and a microfilament inhibitor, cytochalasin B, blocked sperm-induced exocytosis. A guanosine 5'-triphosphate-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, microfilament, and guanosine 5'-triphosphate-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying exocytosis in living eggs.

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