188 



Embryogenesis: Preparatory Phases 



that normally do not give an agglutination 

 reaction, by the addition of a nonspecific 

 adjuvant (from hen's egg white and other 

 sources) along with the egg water. The ad- 

 juvant alone has no agglutinating action 

 on the sperm. Thus the failure of agglutina- 

 tion to occur ordinarily in certain species of 

 animals is not necessarily indicative of the 

 absence of fertilizin in the egg water but 

 can be interpreted on the basis of a uni- 



Table 11. Method of Preparation of Sea 

 Urchin Fertilizin {from Tyler, 

 '48a) 



1. Extract 20% suspension of washed eggs in sea 



water at pH 3.5. 



2. Decant supernatant (agglutination titer = ca. 



1000), centrifuge or filter. 



3. Add 40 ml. N/1 NaOH per liter of supernatant 



(fertilizin precipitates with the Ca and Mg salts 

 of sea water). 



4. Suspend precipitate in 3.3% NaCl, neutralize, 



dialyze. 



5. Remove insoluble particles and precipitate with 



1 /^ to 1 3^ volumes 95% alcohol. 



6. Wash with alcohol and dry (re-precipitate with 



alcohol or saturated (NH4)2S04). (Yield = ca. 

 250 mg. /liter) 



valent condition of either the fertilizin or the 

 sperm. 



A parallel to univalent fertilizin can be 

 found in the field of immunology in recent 

 studies on Rh antibodies. In most cases these 

 antibodies are found to occur in a nonag- 

 glutinating, univalent form (Wiener, '44; 

 Race, '44; Fisk and Morrow, '45; Levine and 

 Walker, '46). Here, too, the addition of an 

 adjuvant (such as serum albumin) enables 

 the antibodies to agglutinate the Rh-positive 

 cells (Wiener, '45a, b; Wiener and Gordon, 

 '48; Diamond and Denton, '45; de Burgh 

 et al., '46). Corresponding to the experimen- 

 tal conversion of fertilizin into a univalent 

 form, immune antibodies have been simi- 

 larly altered by treatment with various 

 agents, such as heat, diazo compounds, for- 

 maldehyde and photo-oxidation (see Tyler, 

 '45a, for references). The alteration appears 

 to involve not simply a splitting of the 

 antibody molecule but also a reassociation 

 of the univalent fragments with fragmented 

 non-antibody protein of the antiserum. Spec- 

 ificity is retained by such imivalent anti- 

 bodies and, in the case of an antitoxin, 

 protective properties persist. The treated 

 antisera are found to have lowered anti- 

 genicity and may, therefore, offer a means 

 of avoiding serum sickness (Tyler, '45a, b; 



Tyler and Swingle, '45). In connection with 

 fertilization, further use has been made of 

 univalent antibodies in experiments with sea 

 urchin sperm (see below). 



A fair amount of information is now avail- 

 able concerning the chemical nature of 

 fertilizin. Lillie ('19) showed that the gelat- 

 inous coat of the sea urchin egg contained 

 large amounts of fertilizin but believed 

 that it was being continuously secreted by 

 the ripe unfertilized egg throughout its 

 functional life. However, more recent ex- 

 periments (Tyler and Fox, '39, '40; Tyler, 

 '40a, '41) have demonstrated that it is iden- 

 tical with material of the gelatinous coat 

 itself and not obtainable from denuded eggs. 

 This has been corroborated by several other 

 workers (Evans et al., '41; Hartmann, '40; 

 Vasseur and Hagstrom, '46; Runnstrom and 

 Lindvall, '46). The presence of fertilizin in 

 ordinary egg water results, then, from the 

 slow dissolution of the coat. For chemical 

 purposes sea urchin fertilizin can be ob- 

 tained in high titer by dissolving the coat 



Table 12. Analysis of Electrophoretically Ho- 

 mogeneous Preparations of Fertiliz- 

 in of the Sea Urchin Strongylocen- 

 trotus purpuratus {from Tyler, '49, 

 and unpublished) 



with dilute acid, without damage to the rest 

 of the egg. With such preparations Tyler 

 and Fox ('39, '40) obtained evidence for 

 the protein nature of fertilizin of the sea 

 urchin and of the keyhole limpet, on the 

 basis of such properties as non-dialyzabil- 

 ity, salting out, common color tests and 

 inactivation with proteolytic enzymes. How- 

 ever, the low values (ca. 5 per cent) ob- 

 tained for the nitrogen content indicated 

 that the material was not a simple protein. 



