Fig. 5. 



Simultaneous measurement of respiration and fluorescence following fertilization of 

 S. purpuratus. Decrease in O2 content is towards the top of the figure. Respiratory rates 

 at various times are indicated on the trace, in 10-1' moles O2 consumed/lO^ cells/sec. 

 Time is from right to left. 



as in Fig. 6. If measured at different amplifi- 

 cations, as in Fig. 8, the timings of the changes 

 were apparently different, acid excretion pre- 

 ceding the fluorescence change. 



The rate of the acid excretion, in eggs of 

 all three species of sea urchin examined, 

 always peaked before the peak respiratory rate 

 (Fig. 7). This suggests that the reactions re- 

 sponsible for the acid formation occur very 

 rapidly, and are essentially over before the 

 respiratory increase. The source and mechan- 

 ism of the acid formation will be discussed 

 later. 



D. Light-scattering changes 



Light-scattering measurements can be a 

 sensitive monitor of structural changes. Ac- 

 cordingly, the kinetics of light-scattering 

 changes following fertilization were measured 

 in collaboration with Dr. B. C. Pressman, 

 using an instrument designed by Dr. Pressman 

 (5). This instrument can simultaneously record 

 all the parameters previously described. 



The results of one such measurement are 

 shown in Fig. 8. It is seen that a light- scatter- 

 ing decrease begins at 45 seconds, and is tem- 

 porally coincident with the beginning of acid 

 excretion. Within five seconds the fluorescence 

 change begins, and this is followed at 60 sec- 

 onds after sperm addition by the activation of 

 respiration. 



These measurements, then, indicate that 

 a temporal differentiation of these events does 

 occur following fertilization. In the remainder 



of this paper, I shall discuss first, the reality 

 and universality of these kinetics, and second, 

 the possible structural and molecular mecha- 

 nisms of the observed changes. 



II. Possible factors influencing the kinetic de- 

 termination 



Several questions can be raised as to the 

 degree the observed temporal sequence reflects 

 the actual sequence. A major biological artifact 

 could be the kinetics of sperm-egg interaction. 

 Thus, if the successful contact between egg and 

 sperm took several seconds or minutes, the 

 timing and duration of the observed changes 

 could simply, and uninterestingly, represent 

 the fertilization time. The experimental condi- 

 tions which would obviate this argument, how- 

 ever, are (1) a large redundancy of sperm 

 were added, and (2) the same kinetics were 

 obtained in the presence of 10-fold less sperm. 



The experimental measurements also pro- 

 vide an estimate of the time for successful 

 sperm-egg interaction, which is related to the 

 duration of that reaction completed in the 

 shortest interval. From Fig. 8, this is seen 

 to be the light-scattering change, which has a 

 % time of only 20 seconds. The % time for 

 fertilization is probably less than this, how- 

 ever, since the light-scattering change in a 

 single cell probably has a finite duration. If 

 this change is identical to that observed in 

 single cells, its duration in one cell would be 

 about 20 seconds (13). 



22 



