200 



PRODUCTION OF DOUBLE EMBRYOS 



B. THE EXPERIMENT: DOUBLE EMBRYOS BY CONSTRICTION 



PURPOSE: To determine the ability of single blastomeres of the 2-call stage to develop 

 complete embryos, follovlng accentuation of the first cleavage furrow. 



MATER IAI£: 



Biolof^ical : Urodele eggs in the 2-cell stage. Amblystoma eggs may be collected in 



nature (see section on Breeding Habits) or Triturus egga may be layed in 

 the laboratory as a result of anterior pituitary stimulation (see Induced 

 Ovulation) . 



Technical : Standard Solution for Anura and Growing Medium for Urodela. 

 0.1^ KOH in appropriate medium. 

 Hair loops, silk fibers, operating glass needles. 



METHOD: 



Precautions : 



1. Avoid over-exposure to the KOH solution (see section on Isolation of Embryonic 

 Cells). 



2. After separating the blastomeres, keep specimen in adequate medium and at a cool 

 temperature. 



Controls : These will consist simply of eggs from the same clutch, kept under identical 

 conditions except for the separation of the blastomeres. 



Procedure : 



Prepare simple loops of fine (blonde) baby's hair so that each loop is slightly 

 greater than the diameter of the egg and its jelly capsule. Prepare several Syracuse 

 dishes with Permoplast base and depressions calculated to hold the 2-cell stage and 

 its Jelly capsule. Fill with appropriate medium and then select eggs in the two 

 cell stage for constriction. 



:^y placing these eggs for a brief period in 0.1^ KOH (made up in the same cul- 

 ture medli i) the surface coat will be weakened and the cleavage furrow will be ac- 

 centuated. Eemove the egg and pass it through three changes of culture medium 

 before the furrow has progressed very far. This should be practiced, for it may not 

 be easy to stop the KOH action as abruptly as desired. 



Constriction of an amplilDiaii egg, 

 wiDiin Its jelly capsule, at the 

 beginning of the 2-cell stage, by 

 means of a hair loop. Wlien blas- 

 tomeres are separated, two embryos 

 develop; when the furrow is merely 

 deepened, double embryos result. 



Remove the 2-cell stage to the Syracuse dish with Permoplast depression and 

 press the hair loop into the bottom of the depression (two ends above the depres- 

 sion) and maneuver the egg into the depression and loop so that the cleavage furrow 

 lies directly parallel to the loop. With practice one can determine whether it is 

 beet to anchor one end of the loop in the nearly Permoplast, leaving but a single 

 loose end to tighten as the egg is held In position by forceps. It may also help to 

 build up the Permoplast about the egg to hold it the better, '/^en secure, use 

 watchmaker's forceps and tighten the loop so that it constricts the 2-cell stage be- 

 tween the blastomeres, through the Jelly capsule and all. It may be necessary to 



