uncoupler of respiration is added to fertilized 

 eggs, there is only a 10-second lag before the 

 increased respiratory rate is evidenced, as 

 compared to a 30-second lag between fluores- 

 cence and respiration when these eggs were 

 initially fertilized. Finally, the lag is evident 

 in other species examined (see, e.g.. Fig. 10), 

 and was also observed by Ohnishi and Sugiyama 

 (14) in several species of Japanese seaurchins. 

 These workers, furthermore, were using a bare 

 platinum electrode with time constants less than 

 one second, as compared to our membrane- 

 covered electrodes with time constants of 3-6 

 seconds. 



The present data, then, indicate that the 

 first discernable event of fertilization - in our 

 measuring system - is a structural change, 

 probably related to cortical granule breakdown 

 (see Sec. IVa). This light-scattering change, 

 observed in cell suspensions, is probably simi- 

 lar to that seen by Rothschild and Swann in 

 single cells under dark field illumination (13). 



Although this structural change occurs 

 early, the first change in the eggs is undoubtedly 

 related to attachment of the sperm acrosomal 

 filament, which probably initiates these struc- 

 tural reactions in a primary, or possibly 

 secondary, reaction. The structural events might 

 also be related to changes in electrical prop- 

 erties of the membrane, as first shown by 

 Tyler et al (15) and Hiramoto (16). The data of 

 Hiramoto is shown in Fig. 9, and indicates an 

 early change in membrane resistance, capaci- 

 tance, and potential upon successful sperm-egg 

 contact. This change precedes membrane ele- 

 vation and might also precede cortical granule 



Time \n minucet 



Fig. 9. 



Data of Hiramoto, showing changes in membrane poten- 

 tial i , membrane resistance i , and membrane capaci- 

 tance A T , following fertilization of Peronella. (Fig. 2, 

 Hiramoto, Exp. Cell Res. 16, 421, 1959; reproduced with 

 permission of Academic Press.) 



' ' ' ' I — I — 1 — I — I — I — I — 1 — I — I — I — I — 1 



-60 60 120 180 240 300 330 360 



Seconds After Sperm Addition 



Fig. 10. 



Respiration rate and extracellular pH following fertiliza- 

 tion of Lytechinus variegatus (data of Epel and Iverson). 



breakdown, although the temporal relationship 

 between granule breakdown and membrane ele- 

 vation is not clearly defined, and might vary in 

 different species (17). 



GROSS: The time is about a minute after 

 fertilization, isn't that right? 



EPEL: Yes. 



MASSARO: Is that from the time of adding 

 the sperm or from the time of contact? 



EPEL: 1 believe it's from the time of sperm 

 addition. However, the important point is that 

 he shows data that indicate relative time of 

 membrane elevation. 



MASSARO: Well, how long does it take the 

 sperm to get in? Where is the sperm after 15 

 seconds? 



EPEL: That's a good question. In some or 

 most organisms an acrosomal filament is ejected 

 from the head of the sperm. In Hydroides this, 

 supposedly, takes place within 9 seconds after 

 you add the sperm. 



POLLARD: Isn't that about where the first 

 indication of change in membrane resistance is 

 seen? The resistance shows quite a change right 

 away. 



EPEL: The best evidence for a rapid 

 change is a change in light-scattering of single 

 cells observed under dark field. This takes place 



24 



