FERTILIZATION AFTER INITIATION OF DEVELOPMENT. 279 



the egg and cause the loss of substances necessary for fertiliza- 

 tion or has caused new chemical combinations to be formed 

 that renders certain substances unavailable, then fertilization is 

 likewise impossible. 



In order to determine definitely whether or not this unfer- 

 tilizable character of the egg is due to a real physiological or 

 physico-chemical condition of the egg substances or whether it 

 is due to physical conditions that prevent the extrance of a 

 spermatozoon into an egg, a large number of these from different 

 kinds of experiments were preserved and sectioned. Usually 

 Boveri's picro-acetic acid was used as a killer and sections (4 ^ 

 thick) were stained in iron haematoxylin. 



IV. OBSERVATIONS FROM PRESERVED MATERIAL. 



The cytological data to be presented is not meant to be a 

 critical analysis of chromosome behavior but is given only to 

 acquaint us with a few of the more general facts of the behavior 

 of sperm in relation to these unfertilizable conditions, and it 

 will be confined to observations upon three different classes of 

 experiments: (i) To mass cultures of eggs that have been exposed 

 to the weaker concentration of hypertonic sea-water for different 

 lengths of time, returned to normal sea-water and immediately 

 inseminated; (2) to mass cultures which have been exposed to 

 the stronger concentration of sea-water for different lengths of 

 time, returned to sea-water and inseminated; (3) to pure cul- 

 tures of eggs that have segmented but once after an exposure 

 to the weaker concentration, and inseminated. 



I. Insemination Following an Exposure to the Weaker Hypertonic 



Solution. 



Experiment: 



2 P.M. Eggs were collected, washed three times. 



2:50 P.M. Transferred to hypertonic sea-water (50 c.c. sea-water + 8 c.c.. 

 2\ m. NaCl). 



Eggs returned at intervals to sea-water, divided into lots A and Ax. Lot A 

 no further treatment; control. 



Lot Ax, immediately inseminated after return to sea-water. Eggs from both 

 lots killed as given in Table III. 



