15 ORGANIZATION AND CELL-LINEAGE OF ASCIDIAN EGG. 
the first (figs. 71-73). The two polar bodies are of approximately the same size, 
and neither ever divides. They are at first composed of clear protoplasm in which 
the chromosomes are free; later the chromatin is dissolved and diffused throughout 
the cell body, so that they stain deeply and uniformly. They may at all times be 
distinguished from the test cells by this staining reaction as well as by their being 
closely attached to or imbedded in the egg. In Crona they may further be distin- 
guished from the test cells by the fact that they are larger than the latter. /т 
many eggs of Cynthia and tn almost all of Crona the polar bodies remain attached 
to or imbedded гп the egg at the point of their formation, and they thus constitute 
a most tmportant landmark. 
В. FERTILIZATION. 
As has been said, the first maturation spindle remains in the metaphase until 
the egg is fertilized. The egg remains capable of fertilization for three or four 
hours at least after the first formation of this spindle. As Castle (1896) has 
shown, self-fertilization rarely if ever occurs in Czona, though artificial cross- 
fertilization is most easily accomplished. In Cyzthza, on the other hand, arti- 
ficial cross-fertilization is successful in only a small proportion of the eggs. 
I have so far been unable to find any artificial means which will cause the 
unfertilized eggs to develop beyond the metaphase of the first maturation division. 
Violent shaking, various degrees of concentration or dilution of sea water, solutions 
of sodium or magnesium chloride of varying strengths have all been without effect 
in this regard. My experience in this matter is similar to that of Lyon (1903), who 
reports that he was unable to cause parthenogenetic development among ascidians 
at Naples by any artificial means. 
1. Entrance of Spermatozoon. 
Of the multitudes of spermatozoa which may be seen burrowing between the 
follicle cells outside of the chorion after spermatozoa have been mixed with the 
ova, only a few ever pass through that membrane. I have never seen a sperma- 
tozoon in process of passing through the chorion and do not know how it is 
accomplished. It is possible that there are one or more micropyles at the lower 
pole, though I have never seen them. In whatever manner the spermatozoa pass 
the chorion it is done very quickly and several frequently enter the perivitelline 
space; dispermy or polyspermy, however, is very unusual. А spermatozoon enters 
the egg in from two to five minutes after the spermatozoa are mixed with the ova, 
and the presence of supernumerary spermatozoa in the perivitelline space is shown 
by the fact that some of the test cells are occasionally fertilized (figs. 80, 85, sn). 
The spermatozoon always enters the egg near the vegetal pole. I have not 
found it possible to determine in living eggs whether the point of entrance lies 
exactly at the vegetal pole or a little to one side of this. In stained preparations 
of entire eggs, as well as in sections, the entering spermatozoon is usually seen to 
lie eccentrically with reference to the vegetal pole (figs. 79, 173). Іп other cases, 
