202 ARTIFICIAL PARTHENOGENESIS AND FERTILIZATION 
an hour and frequently shaken up, the fluid then proved effective. 
Of course, I did not use it directly, but first filtered it five to six times 
through triple filter paper, and then added the salts obtained by the 
evaporation of sea-water so as to make it of the same concentration 
as that of normal sea-water (about 4 per cent). When put into this 
water, unfertilized eggs of both Sphaerechinus and Arbacia showed 
signs of cleavage—each, of course, only responding to the extract of 
sperm of the same species. I may, however, remark at the same time 
that not a very large number and by no means all of the eggs responded; 
it was usually the case that the eggs of one individual would react to 
one and the same sperm fluid, but not those of another. In the best 
cases the segmentation proceeded regularly to the four-cell stage, but 
afterward it became abnormal and the blastomeres which were very 
dissimilar in size fell apart from one another, probably as the result of 
the absence of the vitelline membrane. The velocity of development 
was much slower in eggs so treated than in normally fertilized ones. 
I have no intention of criticizing Winkler’s efforts; they 
were certainly a step in the right direction. But it must be 
pointed out that the experiments are not free from objection. 
In the first place, his sea-water was made much more alkaline 
than is normal, owing to the fact that by first evaporating it 
he drove out the CO, and converted the bicarbonate into car- 
bonate. He afterward restored the sea-water to its normal vol- 
ume by adding distilled water. The increase of alkalinity thus 
produced will alone lead to such results as Winkler describes, 
as I showed years ago. 
For several years I have tried in vain to repeat Winkler’s 
experiments; results such as he described can be obtained 
by using slightly hypertonic or hyperalkaline sea-water, or 
the two together; but these results are also obtained when no 
sperm is added to such sea-water. Before the appearance of 
Winkler’s paper I myself had examined the effect of various 
enzymes upon the unfertilized sea-urchin egg, but with negative 
results. At my suggestion, Professor W. J. Gies, of Columbia 
University, undertook a series of experiments in which he 
subjected the spermatozoa to every known method that leads 
