120 



100- 



80- 



E 

 2 60- 

 'o 



40- 



20- 



DPN 



TPN 

 ■ ■ . 



I I I I I I I I I 

 20 40 60 80 100 120 140 160 180 

 Seconds After Sperm Addition 



Fig. U. 



Analysis of DPN and TPN following fertilization of L. 

 variegatus. Arrows indicate initiation of acid excretion and 

 increased respiration (data of Epel and Iverson). 



S, purpuratus), the acid excretion has begun 

 at 18 seconds after sperm addition. The res- 

 piratory lag is longer here, O2 consumption not 

 increasing until 30 seconds after the pH increase. 



Figure 11 shows that the DPN decrease 

 similarly occurs, beginning after acid excretion 

 and before respiratory activation. Although TPN 

 does not change (analogous to the sea urchin 

 Arbacia punctulata), TPNH does increase (data 

 from separate experiments not shown here). 



A similar temporal sequence was also ob- 

 served in Lytechinus pictus, where measure- 

 ments were done in the Pressman apparatus 

 as in Fig. 8. It thus appears from an examina- 

 tion of two genera and three species, that the 

 temporal sequence is identical as regards 

 changes in structure, fertilization acid, fluo- 

 rescence, and respiration. 



IV. Significance and mechanism of observed 

 changes 



A. Light-scattering and acidity changes 



The observed decrease in light-scattering 

 suggested a volume or size increase. Although 

 the volume of the egg supposedly does not 

 change, there does occur an elevation of a 

 "fertilization membrane". This membrane, in 



the unfertilized egg, lies closely apposed to a 

 peripheral ring of granules - the cortical gran- 

 ules - which rupture upon fertilization, releas- 

 ing their mucopolysaccharide contents. The 

 overlying membrane is then presumably pushed 

 out, or elevated, either by expansion of the 

 mucopolysaccharide through hydration, through 

 osmotic forces resulting from these substances, 

 or molecular unfolding of the precursor mem- 

 brane (see 19). At any rate, the effective volume 

 of the egg doubles, which makes this change a 

 prime suspect as the cause of the light-scatter- 

 ing change. 



This hypothesis can be tested, since the 

 precursor membrane can be removed with 

 trypsin. When this was done - to our great 

 surprise -the identical light- scattering change 

 was still observed. The scattering change, 

 therefore, does not result from elevation of the 

 fertilization membrane. The two most plausible 

 alternatives are that the scattering change 

 represents either the breakdown of the cortical 

 granules (which are trypsin-insensitive), or an 

 actual change in cytoplasmic structure. The 

 latter interpretation is suggested by changes in 

 texture and granularity of the cytoplasm, which 

 can be seen in stained eggs (20) or in vivo in 

 extremely transparent eggs (21). 



That the change might correspond to break- 

 down of the cortical granules is suggested by 

 the similar kinetics of the acid excretion and 

 light-scattering. Although we had initially 

 thought the acid resulted from accumulation of 

 some acidic carbohydrate compound (such as 

 lactic acid), no compound analyzed was present 

 in sufficient concentration to account for the 

 acidity change. This was true for lactate, 

 pyruvate, glucose-6-phosphate, 6-phosphoglu- 

 conic acid, isocitrate, and malate. In fact, the 

 only change so far described which can account 

 for the acid production is the acidic mucopoly- 

 saccharide released by the cortical granules 

 (22). If one assumes that the sulfate moiety of 

 the mucopolysaccharide exists as sulfuric or 

 bisulfuric acid in the granules, then the amount 

 of protons released upon rupture of the granules 

 would be in the same range as the observed 

 acid release after fertilization (23). Although 

 not yet proven, the similar stoichiometry and 

 kinetics strongly support the conclusion that the 

 light-scattering and acid increase result from 

 the same event - the cortical granule break- 

 down. 



Irrespective of interpretation, the kinetic 

 analysis of the light-scattering changes suggests 

 that structural changes may be highly critical 

 in metabolic activation, since they are one of 



26 



