with eggs of other invertebrates suggest that the 

 acrosome region of a spermatozoon is active during 

 the first stages of fertihzation and that it carries 

 a lysine which facilitates the attachment of the 

 spermatozoon to the egg membrane and its pene- 

 tration into the cytoplasm. 



The old view that spermatozoa penetrate the 

 egg by a mechanical action of screw-borer move- 

 ments of the pointed end (the perforatorium) has 

 been abandoned. It is now generally accepted 

 that the action of the sperm head is primarily 

 chemical and that probably several enzymes are 

 carried by the acrosome. Readers interested in 

 the problem of fertilization are referred to compre- 

 hensive reviews of this subject by Runnstrom, 

 Hagstrom, and Perlmann (1959), Colwin, A. L., 

 and L. H. Colwin (1961a, 1961b), and Colwin, 

 L. H., and A. L. Colwin (1961). 



FERTILIZATION OF EGG 



Eggs for fertilization experiments may be ob- 

 tained in the laboratory by stimulating a single 

 female spawn as described in chapter XIV. A 

 suspension of eggs pipetted off the bottom of a 

 laboratory tank is free of blood and other body 

 fluids. Eggs may also be taken directly from the 

 ovary by cutting off smsxll slices from the surface 

 of the gonad and mincing or shaking them in sea 

 water. Cutting into the underlying layer of 

 digestive diverticula should be avoided to prevent 

 contamination with body fluids. The eggs must 

 be washed se^ eral times in filtered seawater by de- 

 canting or by filtration through a fine sieve until 

 the suspension is free of tissue cells and debris. 

 After being in sea water for a short time, the eggs 

 change their shape and become globular but their 

 large germinal vesicles remain clearly visible 

 (fig. 316). 



A sperm suspension may be obtained by any 

 one of three methods. Male spawning can be 

 induced by raising the water temper atiu-e or by 

 adding a small amount of thyroid suspension, and 

 live spermatozoa collected as they are discharged 

 through the cloaca; small pieces of ripe spermary 

 can be e.xcised and the spermatozoa liberated in 

 sea water by shaking; or a very ripe spermary 

 can be pressed gently with the fingertip and the 

 spermatic fluid pipetted as it comes from the 

 gonoduct. Concentrated sperm suspension must 

 be diluted for fertilization. I found it convenient 

 to make a standard suspension using 0.2 g. of 

 gonad material in 50 ml. of sea water and then 



Microns 



30 



Figure 316. — Camera lucida drawing of naturally spawned 

 but unfertilized egg of C. virginica. 



diluting it, using 0.5 ml. for 100 or 150 ml. of 

 water containing eggs. 



Although several spermatozoa may attach 

 themselves to an egg, (fig. 317), only one pene- 

 trates the cytoplasm. The others, called super- 

 numeraries, eventually are cast off when cleavage 



Microns 



25 



Figure 317. — Photomicrograph of a fertilized egg of C. 

 virginica a few minutes after the formation of the 

 fertilization membrane. Several spermatozoa are at- 

 tached to the egg membrane but only one will penetrate 

 it. The germinal vesicle is intact. Contrast phase oil 

 immersion lens. 



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FISH AND WILDLIFE SERVICE 



