ANDROGENESIS l8l 



a few of the egga from the female into one of the Syracuse dishes. At 10 minute Intervals 

 strip additional eggs into other Syracuse dishes, marking the exact time of insemination 

 on each dish. Within 3 to 5 minutes after insemination, flood each dish with Spring Water 

 (or Standard Solution) so that the eggs are completely covered. The other dishes are to 

 be similarly flooded at comparable intervals thereafter. The water on the eggs may be 

 changed to clear it of excess spermatozoa. 



The nucleus is to be pulled out by means of a glass needle. This needle should be 

 made of soft glass but must have a sharp and rigid point. A large number of needles should 

 be made available with an appropriate needle holder. 



When the egg is inseminated the first polar body is already located between the egg 

 and the vitelline membrane. This small gray bead representing an extruded nucleus may 

 sometimes be located on the animal pole surface, near its center. One must focus very 

 sharply onto this animal pole surface, and use light coming onto the egg from an angle, 

 in order to see the very small polar body and its shadow. 



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 depres- 

 sion. This is caused by a temporary retraction of the surface coating just above the form- 

 ing second maturation spindle. 



Insert the tip end of a glaea 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 k Syra- 

 cuse 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 IH THE UROCELE EGG 



The eggs of Triton, Triturxis pyrrhogaster and T. virldescens 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 Breed- 

 ing). 



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

 Important (when using Urodele eggs) to note the exact time of ovlposition of each egg. 

 The removal of the maturation spindle must occur within 50 minutes after ovlposition. 



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 Solutior\ or Spring 

 Water as the medium. 



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

 above. Another method, developed by Xaylor (1957), Involves sucking out the nuclear ele- 

 ments with a mlcro-plpette. The egg must be oriented with the animal hemisphere dorsal 

 in position. Then, with a fine glass needle (1 to 2 ji in thickness) rupture the vitelline 

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

 tion 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.l6 mm. Place the end of the rub- 

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

 adjust 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 



