ARTIFICIAL ACTIVATION ARTIFICIAL ACTIVATION11/3/1999
What does it mean to start development in a sea urchin without using sperm? What about the missing DNA? The missing centrosome?
There are many ways to activate eggs. Two will be mentioned here. One of the easiest is the "Soda Pop" method used by Dr. Daniel Mazia. The other most common method is to use Ammonium Chloride. See also 100 Years Ago, Jacques Loeb
Make up 2 molar stocks of potassium chloride (14.9g/100ml), magnesium chloride*6H2O (40.6g/100ml), magnesium sulfate*7H2O (49.3g/100ml) and calcium chloride*2H2O (29.4g/100ml). In a 150ml beaker add the following in order shown:
|
Potassium chloride, 2M |
0.5 ml |
|
Magnesium chloride, 2M |
1.2 ml |
|
Magnesium sulfate, 2M |
1.2 ml |
|
Calcium chloride, 2M |
0.5 ml |
Just before use add 2.5 grams of sodium chloride and then quickly add 100 ml of sodium free Canada Dry Seltzer water (at temperature of experiment). Immediately add to the eggs that have had the seawater just removed (not completely dry, just lets eggs settle and pour off the seawater).
Let the eggs sit in this media for 1 minute, then slowly pour them into a large volume (one-liter) of normal seawater and slowly let them settle. Capture a few eggs with a pipet/eyedropper and view under a microscope to see the fertilization envelope rise.
WHAT HAPPENS:
The pH of the eggs is lowered by the effects of the weak acid, carbonic acid (CO2 dissolved in water). At the low pH the calcium sequestering machinery of the eggs is shut down and calcium ions are released into the cytoplasm. When the eggs are put back into normal seawater the internal pH of the eggs quickly rises back to the normal pH (sometimes even over shoots then settles down again). The pH rise is too quick for the calcium sequestering machinery to remove the excess calcium ions from the cytoplasm before they can activate the eggs.
This is an easier, but different method. There are two ways to activate with ammonia.
15mM Ammonium Chloride in seawater at pH 8.0 (normal pH of seawater)
1.5mM Ammonium Chloride in seawater at pH 9.0
They give slightly different results as so it is a good idea to try both.
WHAT HAPPENS:
Ammonia is a weak base. At physiological pH it is dissociated into two species:
NH4Cl <-> NH4+ and OH-
In its uncharged form NH4Cl it is able to pass the plasma membrane. Thereupon it re-equilibrates with in the inside of the egg. The pH outside of the egg is normally pH 8.0. The pH inside the egg is normally pH 7.0. The addition of the ammonia raises the internal pH of the egg to around 7.6 -7.8 or near the pH of the fertilized egg. Some researchers have shown that there may be a small calcium release with ammonia used in normal seawater, so you may want to try this in normal seawater and in zero calcium seawater, see Seawater Formulations.
Of course the ideas in this Extended Research project can be combined with other ideas. For instance, what happens when you activate in the absence of sodium ions?