STUDIES OF FERTILIZATION 373 
the vitellne membrane and enter the egg until at least forty to 
fifty minutes after insemination, although its attachment to the 
membrane takes place immediately. (3) That the presence of 
the sperm-nucleus is readily demonstrable in all stages after 
penetration. 
B. Removal of the spermatozoon after membrane formation 
In the summer of 1909 I was studying the effects of centri- 
fuging on the egg of Nereis with the aim of getting more data 
on the problem of polarity and the theory of formative stuffs. 
It soon became apparent that the effects of centrifuging varied 
with the stage of development, and so several series of experi- 
ments were made in which the eggs were centrifuged at regular 
intervals from before fertilization up to the time of the first 
cleavage. 
The effects of centrifuging may be divided into three cate- 
gories: (1) A certain proportion of centrifuged eggs develop 
approximately normally, the percentage varying greatly with 
the stage of centrifuging. (2) A certain proportion of eggs, 
varying at different stages, segment more or less abnormally, 
sometimes extremely so (e.g. meroblastic), and produce embryos 
with more or less pronounced abnormalities. (3) At certain 
stages of centrifuging a variable proportion of eggs fails to carry 
out even the first cleavage. The investigation of the causes of 
such failure to segment revealed the fact that it was owing to 
the removal of the spermatozoon after membrane-formation. 
It is the evidence for this statement that is now under considera- 
tion. 
The results with reference to failure to segment were, in gen- 
eral, as follows: 
1. If unfertilized eggs were centrifuged and then fertilized, 
all segmented, and a large percentage tended to develop quite 
normally. 
2. A disturbing factor comes in shortly after insemination, 
owing to the fact that when the jelly is first secreted by the eggs 
it is so viscid that the eggs stick together in the bottom of the 
centrifuge in a mass which cannot be separated into its constitu- 
