THE EFFECT OF ACE OF THE ECC ON 

 EMBRYONIC DEVELOPMENT 



PURPOSE : To determine the effect on early development of the ageing of the amphibian egg 

 prior to inaemlnatlon. 



MATERIALS : 



Biological : Ovulating females of Eana plplens, sexually mature males. 



Technical : Controlled temperatures at 10°C*. and 20°C. 



METHOD: 



Precautions : The ovulating females must he kept moist and at the constant temperatures 

 designated. 



Control : Eggs fertilized within 2k hours of the time the first ones reach the uteri 

 may be considered as the control eggs. 



Procedure : 



1. Inject two freshly captured, sexually mature females of Rana pipiens each with 

 the pituitary glands from 5 or 6 adult female frogs (or from twice as many male 

 frogs). Place one at laboratory temperatures, the other at 10°C. 



2. Beginning 2k hours after the pituitary injection, test each female by gentle 

 stripping to determine when the first eggs reach the uteri. (Expect the frog 



at the laboratory temperature to have uterine eggs within 2l+-ij-8 hours, the other 

 within i+ to 5 days.) 



5. As soon as eggs appear in the uteri and are easily expressed from the cloaca, 

 consider this the initial time in the ageing process of the eggs. Fertilize 

 200-300 eggs from each female immediately. These will be considered the control 

 eggs. Determine the percentage cleavage and separate the dividing eggs into ap- 

 propriate receptacles with adequate surface and 2 cc. of fluid per egg. Keep 

 them at laboratory temperatures, not higher than 23°C. 



POLAR BODY PIT MARGIN OF 



DEPIGMENTED CORTEX 



DEPIGMENTATION OF ANIMAL HEMISPHERE OF AGED EGG 



Second polar bodj elimination 20 minutes after insemi- 

 nation of frog's (R. plplens) egg at 23° - 2o° C. 



-150- 



