STUDIES OF FERTILIZATION 553 



after violent shaking which removed the jelly and in which the 

 agglutinating power had been reduced from 400 to 10. It would 

 appear from this that it makes no difference how rapidly we dis- 

 pose of the fertiUzin; the eggs are no longer capable of fertili- 

 zation when it is gone. 



b. Cessation of fertilizin production after fertilization 



In a previous paper (Lillie '13) I noted that eggs of Nereis 

 cease to produce sperm-agglutinating substance soon after fertil- 

 ization, but that apparently this was not the case in Arbacia. 

 I, however, ventured the suggestion that the apparent continued 

 production of the sperm-agglutinating substance in Arbacia 

 might be due to the presence of unfertilized eggs, of which there 

 was a considerable percentage in the cultures. This may have 

 had something to do with the results there described, but the 

 factor that leads to an apparent continuation of fertilizin pro- 

 duction in fertilized eggs is its presence in high concentration in 

 the jelly, a condition that I, by no means, realized at first. When 

 this factor was eliminated it was easy to demonstrate that fertil- 

 ized eggs no longer produce the substance. The crucial experi- 

 ments were as follows : 



Experiment 1: August 23, 1913: A-large quantity of unusually fine 

 eggs were washed three times and fertilized at 2:30 p.m. with consider- 

 able excess of sperm. At 2:40 p.m. microscopical observation showed 

 that nearly all had beautifully formed fertilization membranes. About 

 10 per cent of the eggs were without jelly (observation in ink). These 

 usually had as fine membranes as the others. 



The eggs were divided in two equal quantities in test tubes A and B. 

 2:50 P.M.: B was shaken 6 times to get rid of the jelly. Examination 

 in ink showed that the jelly was removed completely from practically 

 all the eggs. A (with jelly left on eggs) and B were then submitted to a 

 series of washings with tests for fertilizin as shown in table 6, p. 554. 



Thus in five washings the B fertiUzed eggs without jelly no 

 longer gave the fertilizin test. This number of washings would 

 be only barely sufficient to remove the agglutinating substance 

 present before fertilization and the loose jelly. Two successive 

 negative tests were given, showing that the production of the 

 agglutinating substance has ceased absolutely. The A eggs were 



