Cortical granule exocytosis in Bufo arenarum oocytes matured in vitro

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 251-259 ◽  
Author(s):  
J. Oterino ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J.N. Valz-Gianinet ◽  
M.I. Bühler
Keyword(s):  
Cortical Granule ◽  
Granule Exocytosis ◽  
Cortical Reaction ◽  
Progesterone Treatment ◽  
Sperm Entry ◽  
Bufo Arenarum ◽  
Chronological Sequence ◽  
Cortical Granule Exocytosis

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy

Zygote ◽  
2006 ◽  
Vol 14 (2) ◽  
pp. 97-106 ◽  
Author(s):  
J. Oterino ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
M.T. Ajmat ◽  
F. Bonilla ◽  
...  
Keyword(s):  
Ultrastructural Changes ◽  
Cortical Granule ◽  
Trypsin Activity ◽  
Trypsin Treatment ◽  
Granule Exocytosis ◽  
Sperm Entry ◽  
Bufo Arenarum ◽  
Vitelline Envelope ◽  
Cortical Granule Exocytosis

SummaryDuring activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

Activation of amphibian oocytes by sperm extracts

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 303-308 ◽  
Author(s):  
F. Bonilla ◽  
M. T. Ajmat ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J. Oterino ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Dna Replication ◽  
Egg Activation ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Granule Exocytosis ◽  
Bufo Arenarum ◽  
Sperm Extract ◽  
Cortical Granule Exocytosis

SummaryIn the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

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.

Using Caged Calcium to Study Sea Urchin Egg Cortical Granule Exocytosis in Vitro

Methods ◽  
1994 ◽  
Vol 6 (1) ◽  
pp. 82-92 ◽  
Author(s):  
Nadeem I. Shafi ◽  
Steven S. Vogel ◽  
Joshua Zimmerberg
Keyword(s):  
Sea Urchin ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Caged Calcium ◽  
Sea Urchin Egg ◽  
Cortical Granule Exocytosis

A comparison of intracellular changes in porcine eggs after fertilization and electroactivation

Development ◽  
1992 ◽  
Vol 115 (4) ◽  
pp. 947-956 ◽  
Author(s):  
F.Z. Sun ◽  
J. Hoyland ◽  
X. Huang ◽  
W. Mason ◽  
R.M. Moor
Keyword(s):  
Transient Increase ◽  
Cortical Granule ◽  
Free Calcium ◽  
Granule Exocytosis ◽  
Sperm Entry ◽  
Sperm Penetration ◽  
Ionic Changes ◽  
Cortical Granule Exocytosis ◽  
Intracellular Changes ◽  
Electrical Stimuli

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after electrostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.

scholarly journals An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis.

1985 ◽  
Vol 100 (4) ◽  
pp. 1325-1329 ◽  
Author(s):  
W B Busa ◽  
R Nuccitelli
Keyword(s):  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Transient Increase ◽  
Cortical Granule ◽  
Entry Site ◽  
Cleavage Division ◽  
Present Evidence ◽  
Granule Exocytosis ◽  
Sperm Entry ◽  
Cortical Granule Exocytosis

The eggs of most or all animals are thought to be activated after fertilization by a transient increase in free cytosolic Ca2+ concentration ([Ca2+]i). We have applied Ca2+-selective microelectrodes to detect such an increase in fertilized eggs of the frog, Xenopus laevis. As observed with an electrode in the animal hemisphere, [Ca2+]i increased from 0.4 to 1.2 microM over the course of 2 min after fertilization, and returned to its original value during the next 10 min. No further changes in [Ca2+]i were detected through the first cleavage division. In eggs impaled with two Ca2+ electrodes, the Ca2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere, propagating at a rate of approximately 10 microns/s across the animal hemisphere. The apparent delay between the start of the fertilization potential and initiation of the Ca2+ wave at the sperm entry site as approximately 1 min. Through these observations describe only the behavior of subcortical [Ca2+]i, we suggest that our data represent the subcortical extension of the cortical Ca2+ wave thought to trigger cortical granule exocytosis, and we present evidence that both the timing and magnitude of the Ca2+ pulse we observed are consistent with this identity. This first quantification of subcortical [Ca2+]i during fertilization indicates that the Ca2+ transient is available to regulate processes (e.g., protein synthesis) in the subcortical cytosol.

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