ANDROCENESIS * 



PURPOSE : To study the variations in early development of the embiyo under the influence 

 of the haploid set of chromosomes from the sperm nucleus alone. 



MATERIAIS : 



Biological : Recently inseminated eggs of any Amphibian, preferably Triturus or Sana. 



Technical : Glass needles and needle holder; micro-pipette (O.16 mm. in diameter) 

 with attached rubber tubing; #2 Stenders with covers. 



METHOD : 



Precautions : 



1. Since the polar bodies are very small and not distinctly colored, it is impor- 

 tant that maxlimim spot-lighting be achieved. The heat of the light must be 

 absorbed, preferably by water-filled Florence flask. The overhead and other 

 lights should be off to reduce extraneous sources of light. 



2. The time of oviposition must be known because the egg nucleus is to be removed 

 as it comes to the surface of the egg to give off the second polar body. In 

 Eana pipiens this occurs from I5 to 55 minutes after insemination. 



5. A minimum of cytoplasm and yolk is to be removed with the egg nucleus. 

 k. Haploid eggs and embryos are less viable than controls, and must be given 

 special post operative care. 



Controls : 



1. Control eggs should be puntured in a manner identical with the experimentals, 

 but at a point well removed from the position of the maturation spindle of the 

 egg nucleus. The same amount of yolk should be removed. 



2 . Some untreated eggs should be allowed to develop to determine whether they are 

 otherwise entirely normal. 



Procedure : 



Provide yourself with optimum lighting conditions. This Includes a spot light 

 directed at the eggs from a ^5° angle in order to shine on the upper surface of each 

 egg and to cast a shadow from the first polar body and, by contrast, to reveal the 

 polar body pit. Low power magnification will be adequate after the polar bodies 

 and pits are recognized. 



It is necessary here to give a brief description of the amphibian egg nucleus 

 at the time of oviposition, and during the few minutes after insemination. The 

 nucleus of the ovarian egg of the hibernating and non-ovulating amphibian is in the 

 germinal vesicle stage, prior to any maturation divisions. This germinal vesicle 

 breakg down at the time of ovulation (liberation from the ovary) so that coelomic 

 eggs show neither a vesicle nor chromosome figures. As the egg enters the oviduct 

 (within 2 hours) the metaphase figure of the first maturation division appears, and 

 as the egg progresses through the upper third of the oviduct it extrudes the first 

 of two polar bodies. The egg nucleus remains near the periphery and about the time 

 the egg reaches the uterus, the metaphase figure of the second maturation division 

 will appear. The egg remains in this condition until it is fertilized (or dies). 

 The procedure described below takes advantage of the peripheral position of the egg 

 nucleus, removing it before it has a chance to fuse with the spemi nucleus entering 

 the egg at another point. 



REMOVAL OF THE FEMALE NUCLEAR ELEMENTS 



An ovulating female frog Is secured and concentrated frog sperm suspensions are pre- 

 pared as thin films In h Syracuse dishes. When the optical equipment is adjusted, strip 



* The author acknowledges, with appreciation, the help of Dr. K. E. Porter In organizing 

 this exercise. 



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