Fig 723The diagram below is a flow chart for egg activation

Fig. 7.23-The diagram below is a flow chart for egg activation in sea urchins. Design experiments to test the following hypotheses. You have at your disposal reagents that block all Ca channels and reagents that will open all Ca channels. You can also make sea water any ionic composition you like. Restoration of of cyclin and mitotic cell cycle Membrane Fast block to Na influx tential polyspermy of MAP kinase Activation of Conversion of Membrane NAD* NAD to NADP biosynthesis change Slow block to nding polyspermy Cortical granule and/or fusion to Ca2+ release egg cell membrane exocytosis Formation of production hyaline layer Phospholipase C Stimulation of Kinase stimulation activation protein synthesis, DNA replication, Diacylglycerol Protein kinase C production activation and cytoplasmic movements of morphogenetic Na /H+ ncrease in exchange intracellular pH material a) Design an experiment to test the hypothesis that Ca release is necessary for the cortical reaction.

Solution

Answer a.

To design an experiment for proof of the hypothesis of role of Calcium in the cortical reaction of Sea urchin, the urchin was injected with Chelating agents. Here EDTA and EGTA are used. Among them, EGTA is more selective towards Calcium than EDTA. After injecting with the chelating agents, then they were allowed to fertilize by addition of sperm. EGTA specifically reduces the subsurface free Calcium concentration to below the threshold for discharge of cortical vesicle, showing that Calcium is very important in Cortical reaction.

Answer c.

Outcome :-

EDTA reduces the normal subsurface free calcium concentration threshold required for cortical reaction. The EGTA, as expected forms a complex with Calcium, thus reducing the Calcium concentration below the reduced value of EDTA. and thus prevents cortical discharge and formation of fertilization membrane. Thus, EGTA specifically reduces the subsurface free Calcium concentration to below the threshold for discharge of cortical vesicle, showing that Calcium is very important in Cortical reaction.

Answer b.

The Calcium influx is important to replenish the Calcium store and this Calcium is used to regulate downstream pathways, i.e. activation of Calmodulin dependent Protein Kinases, thus slow blocking of polyspermy. To prove this hypothesis, the experiment can be designed in the following way -

Incubating ICSI treated (intracytoplasmic sperm injection) eggs in Hepes buffered saline solution containing Gadolinium, this method is known as calcium insulation. This is responsible for inhibiting calcium influx by inhibiting all the channels, depending on the concentration of Gadolinium. Gadolinium is rare earth metal.

Answer d.

Outcome :

With the calcium insulation technique it was observed that only intracellular calcium is not enough for activation of eggs, indeed, it requires extracellular calcium So calcium influx via Gadolinium sensitive plasma membrane channels are required for slow blocking of polyspermy and egg activation.