ANDROGENESIS 181 



From 7 to 10 minutes after insemination there will appear a small depigmented area near 

 the center of the animal pole, and within this area will develop a pin-point depression. 

 This is caused by a temporary retraction of the surface coating just above the forming 

 second maturation spindle. 



Insert the tip end of a glass needle just below this polar body depression at such an angle 

 that it will extend below the spindle and with a very slight withdrawing and upward motion, 

 bring the spindle out with an exudation mass of yolk. This mass will necessarily be 

 somewhat larger than the size of the first polar body, but with practice its size may be 

 reduced and yet include the whole second spindle. Since there are a total of 4 Syracuse 

 dishes of eggs which were inseminated at 10 minute intervals, the second dish will be 

 ready about the time the first dish of eggs has been experimentally treated. 



ANDROGENESIS IN THE URODELE EGG 



The eggs of Triton, Triturus pyrrhogaster and T. viridescens have been used success- 

 fully in androgenesis. The salamander egg is generally fertilized as it passes through 

 the genital tract of the female where spermatophores are stored for variable periods. 

 Ovulation can be induced by anterior pituitary injection (see section on Induced Breeding). 



Since the eggs are fertilized shortly before they are deposited by the female, it is im- 

 portant (when using Urodele eggs) to note the exact time of oviposition of each egg. The 

 removal of the maturation spindle must occur within 30 minutes after oviposition. 



Urodele eggs are normally polyspermic and the multiple sperm entrance points can be 

 identified by dark spots caused representing the accumulation of pigment. Toward the 

 center of the animal hemisphere will be seen a clear area, considerably larger than a 

 sperm entrance spot, marking the position of the metaphase spindle. With watchmaker's 

 forceps remove the several layers of jelly but avoid the vitelline membrane. With a wide- 

 mouthed pipette transfer the egg to a Syracuse (operating) dish in which there is a wax 

 depression appropriately molded to fit the egg. Use Urodele Growing Solution or Spring 

 Water as the medium. 



The Urodele maturation spindle may be removed by the needle method, as described 

 above. Another method, developed by Kaylor (1937), involves sucking out the nuclear 

 elements with a micro-pipette. The egg must be oriented with the animal hemisphere 

 dorsal in position. Then, with a fine glass needle (1 to 2 ^i. in thickness) rupture the vitel- 

 line membrane, but avoid injury to the egg cortex, at several points directly above the 

 position of the spindle. Attach a micro-pipette to a small bore rubber tubing, the tapered 

 end of the pipette having a diameter of not more than 0. 16 mm. Place the end of the rub- 

 ber tubing in your mouth, hold the pipette firmly in one hand, and with the other hand ad- 

 just the low power microscope and the operating dish. Bring the open end of the micro- 

 pipette down directly onto the vitelline membrane just above the region of the spindle and 

 with negative pressure (gentle suction) draw the entire spindle out of the egg. A small 

 amount of cytoplasm and yolk will be included, and this must be kept at a minimum. 

 Transfer the operated egg to a covered #2 Stender with fresh Urodele Growing Solution or 

 Spring Water and keep it at a constant temperature in the vicinity of 15° to 1 8°C. 



CARE OF MATERIAL: 



Operated eggs should be examined within several hours to determine whether they are 

 cleaving. Such eggs as seem to be developing, and show operation exudates, should be 

 isolated in #2 Stenders with the appropriate medium and should be kept at the cooler tem- 

 peratures within the normal range. Controls must be kept at the same temperatures and 

 under the same conditions of medium and space. 



