the first observable changes. If they indeed do 

 represent cortical granule breakdown, the hy- 

 potheses of Moser (24) and Runnstrom and 

 Immers (25), relating granule breakdown to 

 metabolic activation, take on added significance. 



B. Respiratory changes 



Although intensively studied since Warburg 

 first observed the dramatic post-fertilization 

 increase in O2 consumption, the operative 

 respiratory control mechanism is still unclear. 

 One possibility, suggested by the work of 

 Chance (26) and Lardy (27), showing respiratory 

 control by phosphate acceptor (ADP), is that 

 fertilization results in increased ATP utiliza- 

 tion and concomitant ADP formation. The in- 

 creased ADP level could then result in the 

 increased respiratory rate. Such a hypothesis 

 is also suggested by the recent finding that sea 

 urchin mitochondria exhibit respiratory con- 

 trol via ADP (28). To check this possibility, 

 eggs were sampled at rapid intervals after 

 fertilization, and analyzed enzymatically for 

 adenine nucleotides. The results of such as- 

 says, shown in Fig. 12, indicate no significant 

 changes in these coenzymes. Most importantly, 

 there are no changes at the time of maximum 

 respiratory activation. Although this suggests 

 that ADP-limited respiration (State 4-State 3 

 transition) is not operative here, it is probable 

 that ADP produced is immediately rephosphory- 

 lated, and that perhaps it is the ADP content in 

 the mitochondrial micro-environment which is 

 critical. 



An alternative possibility accounting for 

 the low respiration rate in the unfertilized egg 

 is that respiration is substrate-limited. If so, 

 the increased respiratory rate following fertili- 

 zation could result from increased availability 

 of respiration-linked substrate [i.e., a State 

 2 -State 3 transition, as defined by Chance and 

 Williams (26)]. Such a mechanism was first 

 suggested by the findings of Aketa et al. (29) 

 that a large increase in the various glycolytic 

 esters, especially glucose-6-P04, had occurred 

 by five minutes after fertilization. 



To check this possibility simultaneous anal- 

 yses of respiration and glucose-6-P04 were 

 carried out. The results of these experiments, 

 shown in Fig. 13, indicate that such an inter- 

 pretation might be tenable. It is seen that in 

 L. variegatus the glucose-6-P04 level does 

 indeed increase, and begins before the activa- 

 tion of respiration. This increase is rapid and 

 large. By six minutes (not shown) it is six 



90 



80 



70 



^ 60 

 o 



■g 50 



to 



I 40 



'o 30 

 20- 

 I0-- 

 



ATP 



ADP 

 AMP 



20 40 60 80 100 120 



Seconds After Sperm Addition 



Fig. 12. 



Adenine nucleotide levels following fertilization of 



S. purpuratus. 



times the unfertilized level. Changes in glucose- 

 6-PO4 are nowhere near as marked in S. 

 purpuratus, however, nor are they so obvi- 

 ously related to the respiratory activation. These 

 differences could suggest that different sub- 

 trates are being utilized in these two species, 

 or that substrate mobilization is not critical to 

 the respiratory activation. It could also mean 

 that the different levels simply reflect differ- 

 ences in relative enzyme activities and rate of 

 flux of the glycolytic substrates. For example, 

 in frog skeletal muscle the glycolytic flux can 

 increase many fold before any increase in glu- 

 cose-6-P04 is seen (30), whereas in rat heart 

 a flux increase is immediately reflected in a 

 glucose-6-P04 increase (31). Since G-6-P is a 

 substrate in flux, as opposed to a coenzyme 

 which can cycle in its various forms, it might 

 therefore be premature to ascribe too much 

 importance to the different glucose-6-phosphate 

 levels. Rather, the comparative results suggest 

 that fertilization does activate substrate mobil- 

 ization in both cases. 



The enzyme(s) responsible for this mobili- 

 zation is still not known. Glycogen phosphorylase 

 is the best candidate, and is indeed present in 

 both fertilized and unfertilized eggs of S. 

 purpuratus. Furthermore, preliminary experi- 

 ments indicate that the activity of this enzyme 

 is sufficient to account for the peak respiratory 

 activity of the fertilized egg. 



POLLARD: Is all this respiration in the 

 mitochondria? 



27 



