21. PRESSURE EFFECTS ON CLEAVAGE 



PURPOSE: To determine the effect of altering the yolk-cytoplasmic axis on cleavage and 

 on the subsequent development of the embryo. Specifically, to attempt to shift the 

 third cleavage from the equatorial to the meridional plane by the application of un- 

 equal pressure. 



MATERIALS: 



Biological: Fertilized eggs of any Amphibian. 



Technical: Petri dishes, glass tubing 2. mm. in diameter. 



METHOD: 



Precautions : 



1. Do not crowd the eggs; allow sufficient medium for appropriate aeration. 



2. Separate and remove the eggs whose cleavage plane has been altered, plac- 

 ing them in §Z Stenders where they may be given special care. 



Controls : Eggs fertilized at the sanne time, from the same female, but not subjected 

 to any pressure. 



Procedure : 



1. Strip some uterine eggs into a sperm suspension in an inverted cover of a 

 Petri dish. Gently shake them so that they spread out into a single layer of 

 eggs. Flood with Spring Water or Standard Solution in 5 minutes. Mark the 



time of insemination on the dish. 



By 2% hours after insemination these eggs should be in the 2-cell stage, and 

 1 hour later (3^ hours after insemination) most of them should be in the four 

 cell stage. The first two cleavages are normally vertical (meridional) and 

 generally bisect each other in the center of the animal pole. The third 

 cleavage is horizontal but slightly above the true equator of the egg, at right 

 angles to both the first and the second cleavages. 



As soon as most of the eggs are in the 4-cell stage, place the bottom of the 

 same Petri dish over the eggs and, while observing them under low power 

 magnification, add water to the upper dish until pressure is exerted on the 

 eggs to such an extent that they are definitely distorted but not ruptured. 

 The bottom of the Petri dish partially filled with water provides the pressure, 

 and this pressure can be controlled by adding or removing water. The dish 

 also acts as a pseudo-lens so that the eggs beneath can be observed directly 

 and at all times. The pressure must be maintained from before the initia- 

 tion of and through the time of the third cleavage of both the experimentals 

 and controls. 



2. A second method of applying pressure is to draw up the eggs with their jelly 

 into glass tubing which has a diameter slightly less than that of both the egg 

 and its jelly. This will be about 2. mm. for Rana pipiens eggs. The eggs 

 should be drawn up by suction at the 4-cell stage, and observed through the 

 side of the tubing, under water. The eggs will be considerably distorted 

 and sketches should be made while the eggs are under pressure and immedi- 

 ately thereafter. 



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