Uncaging of InsP₃ during maturation.

It has long been known that InsP₃ releases much more Ca²⁺ at the end of the maturation process (for original work on starfish see Chiba et al., 1990; for review see also Carroll, 1996), but no systematic studies have so far been performed on the spatio-temporal aspects of the phenomenon. We have found that the sensitivity of starfish oocytes to InsP3 starts from the animal pole where the germinal vesicle is located and gradually propagates toward the vegetal pole.
To investigate this phenomenon preinjected caged InsP3 was photoliberated at different timepoints during the maturation process.

Procedure:
1. Prepare a mixture of 5mg/ml OGBD 70 kDa with 5-10 uM caged InsP₃.
2. Microinject about 40 immature oocytes with the mixture of OGBD+InsP₃.
3. Prepare acquisition chambers and put injected oocytes into the chambers (a couple of oocytes for each timepoint).
4. Get ready the acquisition setup for experiment.
5. Perform uncaging of an immature oocyte.
6. Add 1-MA and repeat uncaging protocol at desired timepoints during maturation using fresh oocytes for each point.

Important!

• Animal-vegetal axis of oocytes must be parallel to the focal plane
• The UV lamp must be perfectly centered

Results:
The figure below represents results of the InsP3 uncaging during maturation of oocytes of Asterina pectinifera.

Direct injection of 25 mM non-caged InsP3 13 minutes after addition of 1-MA confirms the inhomogeneous pattern of the sensitization to InsP3.