STUDIES OF FERTILIZATION 553 



contain substances from various tissues. But it invariably 

 proved perfectly neutral to spermatozoa of Arbacia, even when 

 taken from females with large ovaries, showing that the substance 

 concerned in agglutination does not escape from the ovaries of 

 'the intact animal, or, if its does, that it is promptly destroyed. 

 Nor was it possible to extract a sperm agglutinating substance 

 for Arbacia by extracting the intestine in sea- or fresh-water.^ 

 These experiments were repeated a sufficient number of times to 

 be conclusive. 



As illustrations: (1) Intestine extractives. August 31, 1912. The 

 intestines of several Arbacia were cut up in about twice their bulk of 

 distilled water, and were allowed to stand in it about an hour. The 

 strongly amber-colored fluid was filtered off and rendered isotonic with 

 the sea-water by addition of concentrated sea-water (four parts of the 

 latter to six of the intestine extract). This fluid causes no agglutination 

 in sperm suspensions. A similar extract of the ovaries caused immediate 

 large dense agglutination masses. (2) Coelomic fluid : As is well known, 

 the coelomic fluid of Arbacia contains large numbers of densely pigmen- 

 ted corpuscles. Outside the body the fluid quickly forms a loose clot 

 which includes many of the corpuscles. The others can be separated 

 from the remaining serum by centrifuging. The corpuscle-free serum 

 was sometimes used, sometimes simply the clot-free serum. Repeated 

 tests were made both by injecting drops into fresh sperm suspensions 

 beneath raised cover glasses, and also by mixing with fresh sperm 

 suspension in vials. Whether the coelomic fluid came from males or 

 females it proved invariably negative, except for the faintest sort of 

 agglutination reaction in one or two cases only, which may have indi- 

 cated some individual differences. It is interesting in view of these 

 facts that outside of the body the coelomic fluid becomes heavily 

 charged with the sperm agglutinin if eggs are placed in it. It must be 

 supposed therefore that the ovarian membrane is impermeable to the 

 agglutinating substance in the intact animal. 



* The experiments this year simply opened up the problems, and it was impossi- 

 ble to make any quantitative tests or adequate chemical examination. For the 

 purpose of the biological problem of the behavior of the spermatozoa with refer- 

 ence to the eggs the question of immediate importance was the behavior with 

 reference to egg-extractives or secretions in the sea-water tested. Stronger agglu- 

 tinating solutions were made by increasing the quantity of eggs with reference to 

 the sea-water, or by crushing the eggs in sea-water. Distilled water was shown to 

 extract more agglutinin from a given bulk of eggs than sea-water; and the coe- 

 lomic fluid of Arbacia also proved to be a better medium for extracting agglutinin 

 from the eggs than sea-water. It would be possible of course to establish quanti- 

 tative values for all of these relations, and this problem should receive attention. 

 The problems of solubility of the agglutinin in various media, and other chemical 

 questions, also present themselves. 



