about 10 seconds after sperm-egg contact is 

 made. 



MASSARO: The sperm is on the outside with 

 the acrosome penetrating? 



EPEL: I don't think there is any direct 

 evidence for that. Certainly the sperm head 

 does penetrate within a short time. However, 

 it takes a relatively long time for it to appear 

 inside the egg. 



GROSS: These things are all cortical 

 changes? 



EPEL: Yes. I doubt if the sperm is con- 

 tributing anything in the initial chemical changes 

 such as genetic information or enzymes getting 

 inside the egg are concerned. As you indicated, 

 the sperm doesn't get in until minutes after. 

 These are surface reactions. 



TS'O: These eggs can only be fertilized 

 by a single sperm? 



EPEL: You can get poly-spermy if you add 

 a very large redundancy, but normally only one 

 sperm penetrates. 



GROSS: The barrier to poly-spermy takes 

 about 20 to 45 seconds to develop at normal 

 temperature. So you'd need a very large multi- 

 plicity. 



EPEL: I think it's more like 10 seconds, 

 although I wouldn't want to say it's that, defi- 

 nitely. [(Added in proof): A short note by 

 Rothschild and Swann (Exp. Cell Res., 2, 137, 

 1951) indicates that the actual block to poly- 

 spermy takes at least 25 seconds, and probably 

 longer. They interpret the failure of the kinetic 

 calculation to apply to the in vivo situation as 

 indicating that the limiting factor is the prob- 

 ability of a "successful" sperm-egg collision.] 



There is one, so far unconfirmed, report 

 which is completely revolutionary. This is a 

 report by Neyfakh etal.(Biochem. Biophys.Res. 

 Comm.18, 582, 1965) on fertilization in fish 

 eggs, which shows that simple contact with 

 sperm is sufficient to activate synthesis of 

 cytochrome oxidase. This activation occurs 

 within one second, and is hence the most rapid 

 change ever reported. 



MAURER: Do we know anything we can do 

 to the sperm which will eliminate this kind of 

 surface contact? 



GROSS: I don't know of any. 



POLLARD: What happens if you ultra- 

 violate the eggs and sperm in vivo ? 



EPEL: They're okay. 



POLLARD: They still do it? 



EPEL: Yes, you can chemically activate 

 the egg without any sperm. 



MAURER: What pushes the button in the 

 sperm? 



EPEL: Presumably interaction between 

 sperm and egg result in ejection of the acroso- 

 mal filament. We have some evidence of in- 

 creases in respiration when you add a very 

 dense sperm suspension. In some cases there is 

 a transient, but definite, increase in respiration 

 (about double). Sperm with no eggs present 

 don't give this. 



TS'O: Anatomically, does the stimulation 

 have to be in the head or tail of the sperm? 



EPEL: Presumably, only the head can 

 stimulate. 



GROSS: The tail never hits first. There's 

 apparently a strong chemo-taxis that orients 

 the sperm in the direction of the egg so that 

 the head goes first. This is important. 



TS'O: Is this because of antibodies? 



GROSS: Well, that's what Tyler says. 

 There's a complicated literature. The assump- 

 tion is that there is a specific receptor in the 

 sperm, and that a product of the egg surface 

 attracts the sperm toward the egg. 



EPEL: There is good evidence for lytic 

 enzymes in the acrosome which may be involved 

 in getting into the egg. Whether these are in- 

 volved in the activation isn't clear. In con- 

 clusion, the in vivo kinetic studies indicate 

 that the timing of structural changes (light- 

 scattering), acid excretion, electrical and fluo- 

 rescence changes (TPNH) cannot at present be 

 temporally separated from each other, but that 

 these can all be temporally distinguished from 

 respiratory activation. This, then, suggests 

 both parallel and cascade-type reactions upon 

 fertilization. 



III. Universality of the temporal sequence 



Because interspecies variations in behavior 

 of other parameters after fertilization of sea 

 urchin eggs have been found (18), itis important 

 to determine whether the above changes occur 

 in other species of sea urchin, and in the same 

 sequence, or whether they are unique to the 

 species so far described. 



Figures 10 and 11 provide a partial answer 

 to this question. The figures depict data, ob- 

 tained in collaboration with Dr. Ray M. Iverson 

 of the University of Miami, on the fertilization 

 changes in the eggs of the sea urchin Lytechinus 

 variegatus. Figure 10, which depicts respiration 

 rate and acid excretion, shows the same tem- 

 poral sequence in these two changes as had 

 ijeen observed in S . purpuratus . Of interest here 

 is the rapidity of the acidity changes. In this 

 species (at 30° C, as compared to 17° C for the 



25 



