12. HETEROPLOIDY INDUCED BY 

 VARIATIONS IN TEMPERATURE 



PURPOSE: To intercede in the kinetic movennents of maturation and amphimixis by utiliz- 

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

 semination, 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 3.0°C., and 34°C. to 37°C. Histo- 

 logical technique equipinent 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 completion of 

 maturation. This means immediately upon egg-laying (for the Urodeles) or 

 within 20 minutes of insemination (for the Anura). 



3. 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 man- 

 ner, 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 

 23°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 tempera- 

 tures is equivalent (and often better) than a long exposure at the lower tempera- 

 tures. 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 ex- 

 periment until recently (Briggs, 1947), partly because their tail-tips do not yield 

 such satisfactory chromosome figures as do the tail-tips of most Urodeles (the 

 Axolotl is the poorest: Fankhauser & Humphrey, 1942). The second polar body 

 is given off from the Anuran egg about 25 to 30 minutes after insemination while 

 it seems to take about 1 hour to emerge from the Urodele egg (Griffith, 1940). 

 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 within 

 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. 



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