170 CARNEGIE INSTITUTION OF WASHINGTON. 



Further, the internal changes occurring between the initial cortical reac- 

 tion and the first division of the egg are not the same in all eggs. 



The species of echinoids which have been used in the investigations are: 

 Strongylocentrotus pulcherrimus, Heliocidaris tuherculata, Clypeaster japonicus, 

 Echinarachnius mirahilis, Temnopleurus toreumaticus, Mespilia globus, 

 Astriclypeus manni, Peronella lesueuri, Toxopneustes pileolus. The recip- 

 rocal crosses were between Heliocidaris and Clypeaster, Temnopleurus, 

 Mespilia, and Astriclypeus. 



Hitherto, the methods commonly used in obtaining cross-activation, 

 when activation was not obtainable in normal sea-water, have been that of 

 allowing the eggs to stand in sea-water for 1 to 4 hours before insemination, 

 or that of increasing the alkalinity of the sea-water by the addition of NaOH. 

 No satisfactory explanation for the success of these methods has been given. 



If it is true that NaOH and such monovalent salts as NaCl increase the 

 permeability of the egg membrane, while such bivalent salts as CaCU and 

 SrCl2 decrease the permeability of the membrane, we have a logical explana- 

 tion of the reason for the success of the methods that I have devised, as well 

 as the explanation of the success of the empirical methods which have been 

 found of use in other cases. 



In some of the cross-activations which I have made, it has been necessary 

 to remove internal block to the union of the egg and sperm nuclei after the 

 cortical block has been overcome. This may be done readily by the use 

 of hypertonic sea-water. 



The following methods proved successful and gave better than 95 per 

 cent activation and cleavage. 



1. Heliocidaris eggs XClypeaster sperms. Inseminated in normal sea-water with exceed- 



ingly good Clypeaster sperm. 

 Clypeaster eggs X Heliocidaris sperms. Inseminated in 100 c.c. sea-water+2 c.c. 

 2.5 MNaCl + 1.2 c.c. N/10 NaOH. 



2. Heliocidaris eggs X Temnopleurus sperms. 



a. Eggsinl00c.c.sea-water-l-2c.c. 2.5 MNaCl. + 1.2c.c. N/10 NaOH for Gminutes, 



then inseminated. 

 6. After 10 minutes tranferred from a to 100 c.c. sea-water +6 c.c. 2.5 MNaCl. 



c. After 20 minutes transferred from b to 82 c.c. sea-water + 18 c.c. 2.5 MNaCl. 



d. After 15 minutes transferred from c to sea-water. 



Temnopleurus eggs X Heliocidaris sperms. Inseminated in 100 c.c. sea-water +1.2 c.c. 

 N/10 NaOH. 



3. Heliocidaris eggs X Mespilia sperm. 



a. Eggs placed in 100 c.c. sea-water+2 c.c. 2.5 M NaCl for 5 minutes then transferred 



to 



b. 100 c.c. sea-water + 1.2 c.c. N/10 NaOH. and inseminated. After 3 minutes eggs 



transferred to 



c. 100 c.c. sea-water+3 c.c. 2.5 M NaCl. After 40 minutes transferred to 



d. Sea-water 



Mespilia eggsX Heliocidaris sperm. Inseminated in 100 c.c. sea-water+1.2 c.c. 

 N/10 NaOH. After 15 minutes transferred to 86 c.c. sea-water + 14 cc. 2.5 M 

 NaCl for 20 minutes, then to normal sea-water. 



4. Heliocidaris eggsX Astriclypeus sperms. Eggs placed in 



a. 0.625 M CaCl2 for 3 minutes. 



b. Then 800 c.c. sea-water +5 c.c. N/10 NaOH+sperms. After 10 minutes to 



c. Sea-water. 



Astriclypeus eggs X Heliocidaris sperms. Eggs placed in 100 c.c. sea-water+2 c. c. 2.5 

 M NaCl. and inseminated. After 10 minutes transferred to sea-water. 



5. Heliocidaris eggsX Peronella sperms. 



a. Eggs placed in 0.625 M CaCl2 for 3 minutes, 



b. Inseminated in 800 c.c. sea-water+5 c.c. N/10 NaOH. 



6. Heliocidaris eggsX Toxopneustes sperms. Eggs inseminated in 100 c.c. sea-water + 



2 c.c. 2.5 MNaCI. + 1.2 c.c. N/10 NaOH. 



