HETEROPLOIDY INDUCED BY VARIATIONS IN 



TEMPERATURE 



PURPOSE: To intercede in the kinetic movements of maturation and amphimixis "bj utilizing 

 extremes (high and low) of temperatures on amphibian eggs immediately after insemina- 

 tion, thereby producing variations in the numbers of chromosome sets within the somatic 

 nuclei (i.e., heteroploidy) . 



MATERIALS : 



Biological : Recently layed eggs of Urodeles; ovulating Anura and mature males of the 

 same and different species. 



Technical : Refrigeration controlled at 0.5°C. to 5.0°C., and 3k°C. to 37°C. Histo- 

 logical technique equipment listed under "Tail Tip Chromosomes". 



METHOD : 



Precautions : 



1. The transfer of eggs to and from the extremes of temperature must be abrupt. 



2. The eggs must be cold (or heat) treated prior to the normal con^letion of matura- 

 tion. This means immediately upon egg-laying (for the Urodeles) or within 20 

 minutes of insemination (for the Anura). 



5. Heteroploids (particularly haploids) are less viable than diploids, and must be 

 given special care. 



Controls : These consist of eggs from the same source, fertilized in the same manner, 

 but kept within the temperature range for normal development. For Urodeles this is 

 generally between 15° and 20°C. and for the Anura between 18°C. and 25°C. 



Procedure : 



The procedure is very simple, but it varies slightly with the different species 

 and the temperature used. In general, a short exposure at the higher temperatures 

 is equivalent (and often better) than a long exposure at the lower temperatures. 

 Triturus viridescens is better than T. pyrrhogaster, which, in turn, is better than 

 the white Axolotl. The Anura have not been used in this type of experiment until 

 recently (Briggs, 19^7), partly because their tall-tips do not yield such satisfac- 

 tory chromosome figures as do the tail- tips of most Urodeles (the Axolotl is the 

 poorest: Fankhauser & Humphrey, 19*^2). The second polar body is given off from the 

 Anuran egg about 25 to 50 minutes after Insemination while it seems to take about 

 1 hour to emerge from the Urodele egg (Griffith, 19'*-0). The extreme of temperature 

 used supposedly suppresses this second polar body formation. 



FOR URODELA 



Urodele eggs are layed singly, and are fertilized as layed by spermatozoa with- 

 in the female genital tract. They should be picked off of the greens (Elodea etc.) 

 and transferred individually to the low (or high) temperature in a marked #2 Stender. 

 After the prescribed exposure, transfer the eggs directly to another container at a 

 temperature of 15° to 20°C. The culture medium is generally Urodele Growing Medium, 

 or Spring Water. Do not crowd the eggs, allowing about 5 cc. of medium per egg. 



FOR ANURA 



Anuran eggs should be secured from an ovulating female, pituitary-induced. The 

 eggs should be Inseminated in the normal manner, flooded within 5 minutes, and trans- 

 ferred abruptly to the low (or high) temperature in Standard Solution or Spring Water. 

 The eggs will stick to the bottom of the container (e.g., finger bowl or Petri dish) 

 and the water may be poured off, and the water of a different temperature added 

 directly. The Anuran eggs need not be separated until after the drastic ten^erature 

 treatment, but at that tine they must be separated into finger bowls containing no 

 more than 25 eggs per 50 cc. of medium, kept at temperatures of 18°C. to 25°C. 



