CLUSTER FORMATION OF SPERMATOZOA 133 



into 50 cc. sea water + 3 cc. tf HCl. The eggs were constantly 

 squirted with a pipette to prevent them from sticking to the glass 

 and were then transferred to normal sea water. They were then 

 washed five times in succession in normal sea water under constant 

 squirting with a pipette and then left standing in a refrigerator 

 with a small volume of sea water. At no time did the sea water 

 in which these eggs were kept give any trace of a cluster formation 

 with fresh sperm. The Supernatant sea water was tested a few 

 hours after the acid treatment and two or three times daily on four 

 consecutive days. These eggs which had apparently lost their 

 chorion had permanently lost the power of giving off to the sea 

 water a substance which causes the cluster formation of the 

 spermatozoa of the same species. If the substance were con- 

 stantly given off by the egg it should have been found after some 

 time in the supernatant sea water. The experiment was repeated 

 a number of times with the same negative result. 



On the other hand, it was easy to show that the acid sea water 

 (50 cc. sea water + 3 cc. ^ HCl) in which the eggs had been 

 washed contained the substance which is responsible for the cluster 

 formation in large quantities. This acid sea water was filtered 

 and the filtrate neutralized with NaOH (with neutral red as an 

 indicator). The neutralized sea water gave with sperm of the 

 same species a very powerful cluster formation. 



This neutralized sea water kept the power of inducing cluster 

 formation for about three days (during which time it stood in the 

 refrigerator) but had lost it the fourth day. 



This seems to indicate that the substance causing cluster forma- 

 tion is derived from the jelly-like substance surrounding the egg 

 (the chorion) but does not emanate from the egg itself. 



If this substance which causes the cluster formation should be 

 identical with the substance which Lillie calls ' fertilizin, ' which is 

 very probable, it is obvious that his conclusion that the substance 

 comes from the egg is untenable. This would also make it im- 

 possible to attribute to this substance a role in the process of 

 artificial parthenogenesis. 



THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 17, NO. 1 



