Effect of Spekm Extract 203 



to the extraction of enzymes from cells. ^ Extracts of spermato- 

 zoa in fresh water, sea-water, alcohol, ether, glycerin, alkalies — 

 all proved absolutely ineffective. The repetition of Winkler's 

 experiments with special attention to the sources of error led to 

 negative results. Gies concluded from these experiments that 

 if the spermatozoon does cause the development of the egg by 

 means of an enzyme, that enzyme either cannot be extracted 

 from the sperm by the usual methods, or it is unable to ent€r 

 the egg. It would also be difficult to imderstahd why Winkler 

 observed no membrane formation if the development really 

 depended upon a sperm extract. 



2. Kupelwieser found in the writer's laboratory at Pacific 

 Grove that if the eggs of the sea-urchin are placed in very con- 

 centrated Mytilus sperm they form a typical fertilization mem- 

 brane in from five to fifteen minutes. The eggs behave just 

 like those in which artificial membrane formation has been 

 produced by a fatty acid. They develop only when subse- 

 quently exposed to hypertonic sea-water, otherwise they dis- 

 integrate. The rapid formation of the fertilization membrane 

 in this case obviously bars the entrance of the spermatozoon 

 into the egg, a process that goes on much more slowly than 

 membrane formation. Kupelwieser succeeded subsequently in 

 obtaining similar results with the filtrate from spermatozoa 

 that had been previously killed by heating to between 70° 

 and 100°. 



I then tried the filtered sperm of Chiton, Asterias, Asterina, 

 S.franciscanus and purpuratm, all of which had been heated to between 

 70° and 100°. In all cases I obtained membrane formation (with the 

 eggs of S. purpuratus). 



The fundamental point about this membrane formation was 



that the concentration of the sperm must be as high as possible. 



It is best to place the eggs directly in the living sperm with little 



or no dilution, for comparison with the dead and filtered sperm that 



1 W. J. Gies, " Do Spermatozoa Contain an Enzyme, Having the Power of 

 Causing tlie Development of Mature Ova ? " Am. Jour. Physiol, VI, 53, 1901. 



