1. The optimum conditions of temperature, volume of sea-water, sur face exposure, and concentration of eggs and of sperm were ascertained.
2. The eggs and sperm used in these experiments were immediately removed from freshly collected sea-urchins.
3. Three species of sea-urchins were used, Toxopneustes and Hipponoe of shallow tropical waters and Arbacia of deeper, colder waters. The fundamental results for all these were in entire accord.
4. The Arbacia germ-cells are less variable than either of the tropical forms.
5. Variability, with respect to cleavage, differs with different individ uals. The eggs of one female when fertilized by a given male may give markedly different percentages of cleaving eggs when fertilized by a second male. The difference is usually much greater, all other condi tions remaining constant, when eggs of different females are fertilized by the same male. The relative difference is usually constant. Some females have a high percentage of cleaving eggs; other females a low percentage. Cleavage is a function of the kind of eggs used.
6. The variations studied were: size and shape of eggs; presence of jelly envelope; membrane formation; cleavage.
7. There is a surprising range of variability for each of these charac ters and characteristics, less for size or shape, greater for jelly and membrane formation, and greatest for cleavage.
8. The eggs of some females showed little variation in size from the norm, were usually globular, and a large proportion had the character istic jelly envelope. These eggs were in good physiologic condition.
9. The eggs of other females varied considerably in size from the norm, had a high percentage of elliptical eggs and a low percentage of intact jelly envelopes. These eggs were in poor physiologic condition.
10. In some females the eggs formed fertilization membranes in about 2 minutes, others required 3 to 6 minutes, or formed no mem branes at all. Rapid membrane formation was associated with small
variability of size, globular shape, and high jelly count; slow formation with enlargement or considerable variation in size, high percentage of elliptical eggs and low jelly count. The former were in good, the latter in poor physiologic condition.
11. The rate of early cleavage and the total cleavage varied most widely. The total cleavage varied from complete sterility to 100 per cent cleavage. High cleavage is correlated with normal size, globular shape, large jelly count, and rapid membrane formation; and vice versa, low cleavage with the opposite conditions.
12. This amazingly large variation is due to: a. A primary, small variability in ripe eggs.
b. The effect of chemico-physical agencies acting on the ripe eggs within the body of the female.
c. A variation in time or ripening of different groups of eggs within a given female.
d. The time between maturation and removal of the eggs to sea-water, which time may be different for different females.
e. The differential effect of sea-water upon physiologically different eggs.
Eggs which have been removed from different individuals at the same time and are of the same chronologic age are not necessarily in the same physiologic condition. The extent of the change may be determined by the study of their variations.
13. For certain experimental work the eggs should be grouped, not according to age—i. e., the time since removal from the body—but according to their physiologic state, which may be accurately deter mined by the various tests suggested above.