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Regulation of the fertilization current in ascidian oocytes by intracellular second messengers
1Laboratory of Cell and Developmental Biology, Stazione Zoologica Anton Dohrn, Naples, Italy.
Abstract:
Neomycin, injected into ascidian oocytes to a final concentration of 10-50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm-activated fertilization currents are not inhibited in the presence of 100 micrograms/ml intracellular heparin, suggesting that these currents are not directly gated by InsP3. The sulfhydryl reagent thimerosal at 100 microM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP3 receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores.
Insights
Phosphoinositide hydrolysis is crucial for ascidian oocyte activation. However, fertilization channels are not calcium-gated from intracellular stores, differing from other cells.
Area of Science:
- Cellular biology
- Developmental biology
- Reproductive biology
Background:
- Fertilization triggers rapid cellular events in oocytes.
- Second messengers like inositol trisphosphate (InsP3) are implicated in oocyte activation.
- The role of intracellular calcium stores in regulating plasma membrane channels during fertilization is debated.
Purpose of the Study:
- To investigate the role of phosphoinositide hydrolysis in ascidian oocyte activation.
- To determine if fertilization currents are gated by InsP3 or intracellular calcium.
- To elucidate the mechanism of plasma membrane channel regulation during fertilization.
Main Methods:
- Microinjection of neomycin to inhibit phosphoinositide hydrolysis.
- Application of intracellular heparin and thimerosal to study fertilization currents.
- Depletion of intracellular calcium stores using EGTA.
Main Results:
- Neomycin inhibited fertilization currents and surface contraction, indicating phosphoinositide hydrolysis is essential.
- Heparin did not inhibit fertilization currents but blocked surface contraction, suggesting InsP3 receptors mediate propagation.
- EGTA-induced calcium depletion did not activate channels, but subsequent fertilization resulted in enhanced currents.
Conclusions:
- Phosphoinositide hydrolysis is required for early activation events in ascidian oocytes.
- InsP3 receptors play a role in propagating the activation response.
- Plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores, unlike in somatic cells.