18 ORGANIZATION AND CELL-LINEAGE OF ASCIDIAN EGG. 



the lirst (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 Ciona they may further be distin- 

 guished from the test cells by the fact that they are larger than the latter. In 

 many eggs of Cynthia and in almost all of Ciona the polar bodies remain attached 

 to or imbedded in the egg at the point of their formation, and they thus constitute 

 a most important landmark. 



B. 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 Ciona, though artificial cross- 

 fertilization is most easily accomplished. In Cynthia, 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. A 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. SO, 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). In other cases, 



