19. ANDROGENESIS 



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

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



MATERIALS: 

 Biological: 



Technical: 



METHOD: 



Precautions: 



Recently inseminated eggs of any Amphibian, preferably Triturus or 

 Rana. 



Glass needles and needle holder; micro-pipette (0. 16 mm. in diameter) 

 with attached rubber tubing; #2 Stenders with covers. 



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

 tant that maximum 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 re- 

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

 In Rana pipiens this occurs from 1 5 to 35 minutes after insemination. 



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



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

 special post operative care. 



Controls: 



1. Control eggs should be punctured 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 45° 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 re- 

 veal 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 breaks 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 ex- 

 trudes 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 sec- 

 ond 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 sperm nucleus entering the egg at another point. 



* The author acknowledges, with appreciation, the help of Dr. K R. Porter in organizing this exercise. 



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