Self -Fertilization Induced by Artificial Means. 173 



different In composition to supply the necessary stimulus. The 

 ether might be supposed to make the sperm sufficiently different 

 from the egg to start the cleavage, or the ether might itself supply 

 the stimulus which is capable of starting the development of the 

 egg after the spermatozoon has entered. 



The test of this view should be found in direct . observation 

 of the eggs themselves. I prepared therefore a series of eggs 

 of Ciona, some unfertilized for check series, others self-fertilized, 

 but not put into ether, and others like the last, but put into ether. 



The difficulties of determining whether the spermatozoa can 

 enter the eggs of the same individual, but fail to start the devel- 

 opment, are greater than may appear at. first sight. The sperm 

 head Is so minute that If after it entered no changes were af- 

 fected In the protoplasm about it, Its presence might be readily 

 overlooked, and since the spermatozoon of Ciona^ enters the egg 

 in a granular zone that colors more deeply in certain stains than 

 does the rest of the egg, the difficulty is thereby increased. Of 

 course I have been on my guard against cases where the sur- 

 rounding sperm have floated over the section, as sometimes hap- 

 pens, or have been carried over it by a defect In the knife, and I 

 have also been careful to exclude all cases where specks of foreign 

 matter may have been on the slide, or in the fixative. There are 

 also two further precautions to be taken. When the egg with- 

 draws from the membrane and the test-cells are extruded, as it 

 were, from the outer zone of the egg, the protoplasm is some- 

 times drawn out in mamiliform processes that stain deeply and 

 resemble the entrance cone formed by the spermatozoon pene- 

 trating certain eggs. Even when the protoplasm does not pro- 

 trude, deeply staining spots are generally present and are espe- 

 cially obvious after Iron haematoxylin. Careful staining with 

 Delafield's haematoxylin shows clearly that these spots have noth- 

 ing to do with the entrance of spermatozoa. Furthermore these 

 spots are found in unfertilized eggs. After iron haematoxylin 

 minute deeply staining bodies, flattened against the outer surface 

 of the egg, can generally be found, and these strongly suggest 

 spermatozoa. That they are not such is shown by their presence 

 in unfertilized eggs, and by their absence after the Delafield 



