STUDIES OF FERTILIZATION 541 



4. SEPARATENESS OF HETERO-AGGLUTINATING SUBSTANCES IN 



EGG-SECRETIONS 



I stated in my previous paper that in strong agglutinating 

 solutions from Arbacia eggs and ovaries there is present also a 

 substance that agglutinates the sperm of Nereis, and I argued 

 that this was probably distinct from the iso-agglutinating sub- 

 stance because (1) it is contained in the blood of Arbacia which 

 is normally entirely free from the iso-agglutinating substance, and 

 (2) it may disappear from agglutinating solutions on standing 

 while the iso-agglutinating substance remains apparently intact. 



Some farther experiments made this year demonstrate the fact 

 that the substance in agglutinating solutions from Arbacia eggs, 

 that attacks Nereis sperm is a separate substance. We may 

 note in the first place that the effect of this substance on the 

 Nereis sperm is distinctly toxic. A drop containing it injected 

 into a Nereis sperm suspension beneath a raised cover slip causes 

 the formation of a permanent coagulated ring at the margin of 

 the drop. In this respect the action differs from the iso-aggluti- 

 nation which is without toxic effects. 



In the second place, the quantity present in strong iso-aggluti- 

 nating solutions of Arbacia is extraordinarily variable; thus on 

 June 18, 1913, an agglutinating solution was made by addition 

 of 3 parts sea-water to 2 parts cut-up ovaries and eggs of Arbacia. 

 The test on Nereis sperm showed: (1) undiluted, formation of 

 ai solid ring-coagulum; (2) diluted one-half with sea-water, solid 

 ring-coagulum; (3) diluted to one-fourth with sea- water, slight 

 permanent coagulum; (4) diluted to one-eighth, small coagulated 

 masses; (5) diluted to one-sixteenth, nearly neutral; (6) diluted 

 to one-twenty-fifth, absolutely neutral. Acting on Arbacia sperm 

 the same solution gave a 5-second reaction at 1/200 dilution. 

 Other samples equally strong in the iso-agglutinating substance 

 were nearly neutral for Nereis sperm undiluted. 



In the third place it is possible to precipitate out all of the 

 Nereis-active substance with Nereis sperm and leave the full 

 complement of iso-agglutinating substance. Thus on June 19 

 an agglutinating solution was made by addition of 3 parts sea- 



