“EFFECT OF SPERM 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 enter 
the egg. It would also be difficult to understand 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 purpuratus, 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 
1W. J. Gies, ‘‘Do Spermatozoa Contain an Enzyme, Having the Power of 
Causing the Development of Mature Ova?” Am. Jour. Physiol., VI, 53, 1901. 
