August 18, 1922] 



SCIENCE 



203 



point for three minutes. To avoid any possi- 

 bility of living sperm being present, the wall 

 of the tubes are thoroughly flamed. The tubes, 

 together with one containing normal sea-water 

 kept at the boiling point for three minutes, are 

 then set aside to be used after from two to 

 twenty-four hours. For an experiment ten 

 drops of each suspension and ten drops of the 

 boiled sea-water are placed in dishes each con- 

 taining one drop of dry eggs. Two minutes 

 later to each dish are added ten cc of sea- 

 water. A control, uninseminated eggs in nor- 

 mal sea-water, was always kept. If the control 

 showed jelly . formation the experiment was 

 discarded. '' 



Uninseminated eggs of Nereis exposed to 

 these boiled sperm suspensions and to boiled 

 sea-water in this way give the following results : 

 boiled sea-water, no development; sperm boiled 

 in sea-water, no effect beyond small per cent, 

 of jelly formation, maturation and differentia- 

 tion without cleavage; sperm boiled in oxalated 

 sea-water, higher per cent, of differentiation 

 without cleavage. The highest per cent, of 

 swimming larvae (differentiation without cleav- 

 age) ever obtained with the NaCl boiled sperm 

 was about 10 per cent.; the highest with oxa- 

 lated sperm was 32 per cent. And this propor- 

 tion usually holds. 



The first experiment of this kind was made 

 early in June, 1914. At Dr. F. R. Lillie's sug- 

 gestion these experiments were repeated directly 

 under his supervision in his laboratory at the 

 Marine Biological Laboratory, Woods Hole, 

 Mass., during each Nereis "run" of 1915. It 

 gives me great pleasure to acknowledge my 

 indebtedness to him for many helpful sugges- 

 tions in this work. 



Every possible precaution was taken against 

 contamination; every variation in procedure to 

 be thought of was tried; and throughout one 

 season, whenever Nereis eggs were to be had, 

 the experiments were studiously repeated. 

 Despite the precautions and the laborious repe- 

 titions, both during 1915 and subsequent sea- 

 sons, it is impossible to reduce the results to 

 any seeming order. Even after the elimination 

 of certain sources of error the results are 

 inconstant. The sources of error may be men- 

 tioned : 



1. Failure to use perfectly fresh eggs of 

 high cortical sensitivity. In 1915 I had great 

 difficulty in keeping the worms from shedding 

 their eggs when kept in the laboratory over 

 night. I therefore adopted the plan of keeping 

 the animals in the refrigerator. But this only 

 made matters worse, for the females shed very 

 quickly on removal from the low temperature. 

 Such eggs were of little value. Moreover, if 

 transfer from the cold sea-water to that at room 

 temperature be suddenly made not only are the 

 eggs shed but they are induced to form jelly 

 and maturate. Moreover, eggs from animals 

 that have been kept at low temperature are not 

 best for study of cortical changes. Such eggs 

 are apt to be polyspermia and in other ways 

 give evidence of change in the cortical reaction. 

 I have found that keeping Cumingia (dry) in 

 the refrigerator markedly changes the normal 

 cortical reactions. The eggs of both forms kept 

 in this way give good cleavage and SAvimmers, 

 but they do not give the best cortical reactions. 

 For the work with spei-m boiled in oxalated 

 sea-water, then, one must have fresh eggs taken 

 from dry females. 



2. The oxalated sea-water should not be more 

 than twenty-four hours old when used. It is 

 best not to use stock solutions. Various experi- 

 ments were made with different molecular solu- 

 tions of the oxalates in distilled water plus the 

 addition of double sea-water to correct the 

 hypotony. But the distilled water solutions 

 seem to deteriorate. It was found best to add 

 from 0.1 to 0.25 grams of the oxalate to 100 

 ee of sea-water which was filtered before use. 

 But even with fresh eggs and the optimum 

 oxalated sea-water the results are far from 

 uniform. 



Since hypertonic sea-water alone initiates 

 development in the egg of Nereis, we might 

 think that boiling the sea-water is alone respon- 

 sible for the results. But boiled sea-water 

 alone has no effect. Since oxalate in sea-water 

 initiates development, we might argue that it 

 is the oxalate in the sea-water rather than the 

 dead sperm that initiates development. How- 

 ever, oxalate alone to call forth development 

 must be present in greater amount than in the 

 suspension of sperm boiled in oxalated sea- 

 water. 



