184 



Embryogenesis: Preparatory Phases 



loss of motility. These experiments of Mac- 

 Leod on human spermatozoa have been con- 

 firmed and extended by Tosic and Walton 

 ('46) and Tosic ('47) with bull spermatozoa. 



In contrast to mammalian sperm, that of 

 sea urchins becomes rapidly immotile under 

 anaerobic conditions, as Harvey ('30) dem- 

 onstrated. Rothschild ('48a, b, c) and Spikes 

 ('48, '49b) have shown that glycolysis does 

 not occur to any very appreciable extent 

 aerobically or anaerobically in presence or 

 absence of glycolyzable sugar. Spikes pre- 

 sents evidence to show that oxidation pro- 

 ceeds through the usual fructose diphosphate, 

 triosephosphate and Krebs cycle pathway. 

 From the spectroscopic identification of cyto- 

 chromes a, a-i, b, c, and COaz (Ball and 

 Meyerhof, '40; Rothschild, '48a) and the 

 demonstration (Rothschild, '48a, c) of photo- 

 reversible CO-inhibition (Fig. 55), it is con- 

 cluded that the respiration of the sea urchin 

 sperm is under control of the cytochrome 

 system. Rothschild ('48c) has also shown 

 that oxidizable substrates and their dehydro- 

 genases are still present in sea urchin sperm 

 that have aged to the point of no motility 

 and respiration. Recent evidence (Rothschild 

 and Cleland, '52) points to phospholipid as 

 the principal endogenous source of energy 

 for motility. From the experiments on pro- 

 longing the duration of motility and fertiliz- 

 ing capacity by means of amino acids and 

 other metal-chelating agents (Tyler, '53) 

 it is also clear that death of the spermatozoa 

 upon dilution in ordinary sea water is not 

 due to exhaustion of their food supply. These 

 agents, evidently by binding toxic trace met- 

 als, enable the sperm to utilize their endog- 

 enous substrate more fully. 



Senescence of Eggs. The unfertilized egg 

 likewise has a relatively limited life-span 

 under ordinary conditions, both in animals 

 with external fertilization and in those with 

 internal fertilization. In certain cases, as in 

 many fish and Amphibia, fertilizability is 

 lost within a few seconds or minutes after 

 deposition in water. In these, marked visible 

 changes, involving elevation of a membrane, 

 are generally noted vipon contact with the 

 new medium and these are very likely in- 

 volved in the loss of fertilizability. A rapid 

 loss of fertilizability may, however, occur 

 without such visible changes, as in the case 

 of Platynereis described by Just C15b, c). 

 In most animals loss of fertilizability and 

 cytolysis of the egg occur in a period of sev- 

 eral hours to one or two days under ordinarv 

 conditions. This relatively rapid senescence 

 cannot be attributed to depletion of endog- 



enous nutrient since, if fertilized, the eggs 

 can survive considerably longer without any 

 added nutrient. For example, fertilized sea 

 urchin eggs will survive for about two weeks 

 without any external source of food, while 

 respiring at a rate that is at least ten times 

 that of the unfertilized egg. The latter might, 

 then, be expected to survive over twenty 

 weeks instead of the two days obtained under 

 ordinary conditions. 



Experiments by Whitaker ('37), Tyler, 

 Ricci, and Horowitz ('38), Tyler and Dessel 

 ('39), Schechter ('37, '41) and others have 

 shown that various agents such as weak 

 alcohol, slight acidity, and low calcium con- 

 tent of the medium can prolong the fer- 

 tilizable life of eggs of marine animals to 

 some extent. It has also been shown (Tyler 

 et al., '38) that sterile conditions extend the 

 survival time of sea urchin eggs by five-fold 

 or more. The deleterious effect of bacteria 

 does not manifest itself until after a certain 

 immune period, since freshly shed eggs can 

 survive in dense bacterial suspensions almost 

 as long as in ordinary sea water. Corre- 

 sponding with the onset of the susceptible 

 period the surface of the egg vmdergoes some 

 disintegrative changes which are manifest 

 by the formation of a tight membrane or no 

 membrane upon fertilization. In the absence 

 of bacteria the eggs remain viable and fer- 

 tilizable for a considerable time after these 

 changes have begun. The agents mentioned 

 above that prolong the functional life of 

 the egg in nonsterile conditions evidently 

 operate by delaying the onset of these auto- 

 lytic changes. 



Runnstrom ('49) has described in some 

 detail the changes that unfertilized eggs of 

 sea iirchins undergo upon aging. Alterations 

 in the surface are detectable by various sorts 

 of tests, such as the hypertonicity test in 

 which, as the eggs shrink, the formation of 

 wrinkles is followed. Ripe unfertilized eggs, 

 shrinking in hypertonic solution, form nu- 

 merous wrinkles on the surface (indicative 

 of a semisolid state), which later smooth 

 out. Eggs which have aged, so that they 

 form low membranes or no membranes upon 

 fertilization, shrink with a smooth surface, 

 indicative of a liquefied state of the surface. 



It seems reasonable to assume that energy 

 is required to prevent the breakdown of the 

 svirface that occurs upon aging. It is of 

 considerable interest, then, that adenosine 

 triphosnhate, at low concentrations (0.002 to 

 0.001 M) has been found (Wicklund, '49, 

 reported by Runnstrom, '49) to be effective 

 in prolonging fertilizable life and even capa- 



