ABSTRACT
Unfertilized sea-urchin eggs produce acid when they are cytolysed or fertilized (Ashbel,, 1929; Runnstr öm, 1933 a, 1935). Neither the post-fertilization nor the post-cytolysis acid has been identified. The number of different acids which might be released is large, and it has not so far been considered expedient to attempt direct analysis of the sea water surrounding the activated eggs. One way of establishing the nature of the acid is by observing the effect of specific metabolic inhibitors on its production. Runnström (1933 b, 1935) used this technique, but the inhibitors used were all negative in their effect, i.e. they did not inhibit acid production. Runnströom investigated the effect of inhibitors which affect one type of glycolysis.1 This paper is also concerned with the effect of a glycogen breakdown inhibitor. If the acid production after cytolysis is associated with the formation of hexose phosphate from glycogen, it should be partly or wholly inhibited in the presence of phlorizin (Lundsgaard, 1933).
The production of a strong acid by a tissue causes a large positive pressure on the tissue side of the manometer and, in a medium containing bicarbonate, the rate of CO2, production may be greater than the rate of CO2, absorption by the KOH.2 It is therefore impossible to measure the O2, consumption immediately after activation or cytolysis by the usual methods, and the estimation of O2, consumption, egg CO2, production, sea-water CO2, production by egg acid, and CO2, retention (Dixon, 1934) by cytolysed eggs at short consecutive time intervals is somewhat complicated. In order to decide if phlorizin inhibits acid production, the presence or absence of the positive pressure on the egg side of the manometer when no KOH is present is a satisfactory indicator provided that certain precautions are taken.
MATERIAL AND METHODS
Ripe unfertilized eggs of Echinus esculentus were obtained either by allowing females to shed into beakers of sea water, or by removing the ovaries and washing their contents through bolting silk. The jelly was removed by four to five washings with sea water over a period of about hr. The eggs were examined for fertilizability when obtained and immediately before each experiment. Cultures which did not show satisfactory activation were rejected. Eggs were left in sea water containing phlorizin, hereafter known as P-water, for at least hr. with repeated washings. Cytolysed eggs were examined microscopically after the experiments.
The experiments were done aerobically in Barcroft differential manometers without KOH. Reagents, except where otherwise stated, were added from small glass tubes hooked on to the KOH cylinders inside the manometer vessels (Keilin, 1929). These are dislodged into the egg suspensions in the manometer vessels by removing the manometers from the water-bath and giving them a tap with the hand. The operation takes about 5 sec. per manometer.
Before each experiment, the pH of the P-water was adjusted to 8.2, the normal value of Millport sea water at this time of year.
In all experiments an approximate M/500 solution of phlorizin in filtered sea water was used.
Cytolysis was effected by saponin, a solution containing 3 g./100 c.c. filtered sea water being used throughout. A given amount of saponin will only cytolyse a given number of eggs. If a suspension of unfertilized eggs is shaken in a manometer in the presence of saponin, four types of eggs may be recovered: (1) eggs which appear normal though they cannot be fertilized, (2) eggs with fertilization membranes and distinct cell membranes in which the cytoplasm appears to be coagulated, (3) eggs without fertilization membranes but with cell membranes within which the cytoplasm is completely disrupted and dispersed, and (4) “eggs” with no fertilization membranes and no cell membranes. The eggs are dispersed in the sea water. As these four effects of saponin might not all be associated with the same amount of acid production a concentration of saponin was used which caused cytolysis either of type 3 or 4 and chiefly 4 in all eggs in the manometer vessels.
The number of eggs in the manometer vessels was estimated by Shapiro’ s (1935) dilution method. This consists in taking a known volume of the egg suspension to be used and diluting it a known amount. This diluted suspension is mechanically agitated until the eggs are uniformly suspended. A sample is then sucked into a capillary tube in which there is a section of known volume. The number of eggs in this section is counted under a binocular microscope and the procedure is repeated twenty times. Egg volumes are measured independently. Results can thus be given per egg or per mm.3 of egg substance.
RESULTS
A typical experiment is shown in Fig. 1, the difference in meniscus levels in the manometer arms being plotted against time. The arrow indicates the time at which saponin was tipped in, this being done after temperature and gas equilibration. One curve shows the acid production of unfertilized eggs cytolysing in ordinary sea water at pH 8.2, the other that of unfertilized eggs cytolysing in P-water at pH 8.2. The curves are scaled according to the number of eggs. The exact shape of the curve at the beginning of the reaction is not important in these experiments and is not to be taken as an exact measure of the rate and quantity of acid production. At the end of the reaction, the curve becomes asymptotic with the x-axis, and if there is a significant difference between the end-point values of control eggs and those in P-water, a difference in the amount of acid produced is indicated provided that certain sources of error are eliminated. The end-point ratios of all comparisons done are shown in Table I. Eggs from different urchins were variable in the amount of acid they produced, though experiments with eggs from the same female were reproducible. [Runnström (1933 a) also noticed this.] Interpretation is therefore only satisfactory when comparisons are made on eggs from the same female. The eggs in each comparison in Table I are from the same female. In each comparison the effect of the P-water in reducing acid production is obvious. In one case, Comparison 9, there appeared to be no acid production at all in the phlorizinized eggs.
The sources of error which have to be eliminated are (1) pH differences in sea water and P-water. This has already been discussed. (2) Buffer effect of phlorizin on sea water. There is no effect. (3) It is not possible to arrange that there is exactly the same number of eggs in each manometer vessel. All results have therefore been scaled so that the ratios in Table I refer to mm.3 of gas per same number of eggs or same amount of egg substance.1 This might introduce a source of error. If there are more eggs in a particular control experiment than in the phlorizin experiment, the controls will produce more acid in any case. The results are therefore not comparable unless the values are scaled according to the number of eggs. This situation is quite satisfactory, but if the P-eggs are present in greater numbers than the control eggs, a complication might arise. The amount of acid produced might appear to be less, even though there were more eggs, owing to the buffer action (“retention”) of the cytolysed egg contents. It is therefore necessary to establish that in a given suspension of cytolysed eggs, the buffer action is not sensibly increased by increasing the concentration of the cytolysed eggs. In the suspensions used a change of 100% in cytolysed egg concentration caused no significant change in buffer action.1 In any experiment the difference in egg concentration was not as much as 100 %. Apart from this, data was obtained which shows that the marked effect of phlorizin in inhibiting acid production is not due to any errors discussed above. In Table II (a), a particular comparison is shown in which there were more eggs in the control than in the P-eggs; (b) is the same comparison scaled according to the number of eggs ; (c) is a particular comparison in which there were less eggs in the control than in the P-eggs; (d) is the same comparison scaled according to the number of eggs. In each case the effect of phlorizin is so marked that the difference between control and P-eggs is evident whether the results are scaled or not, and whether controls or P-eggs have the higher egg concentrations.
DISCUSSION
Although Runnström (1935) has suggested that the acid production which occurs during cytolysis is similar to that produced during sperm activation, there are no a priori grounds for accepting this suggestion, in spite of Loeb’ s well-known Lysin theory (1914) of fertilization and parthenogenesis. There are, in fact, reasons for not believing that this is the case. First, the “acid of injury” is of much more general incidence among cells than the capacity for fertilization. Secondly, if unfertilized eggs are placed in weak hypertonic sea water, acid formation occurs but the eggs can be fertilized afterwards. If fertilization is irreversible, and if fertilization cannot be superimposed on parthenogenetic activation, two concepts which are usually accepted,2 the production of acid by eggs in weak hypertonic sea water can scarcely be considered as parthenogenesis. Sufficient has been said to indicate that the interpretation of the behaviour of cytolysing eggs must at present be treated separately.
Runnström’ s method of cytolysing sea-urchin eggs was by freezing and thawing (1935). The acid produced during this treatment was not affected by 0.03 M iodoacetate 0.06 M fluoride or hexose monophosphate. This result appears to exclude the possibility of the acid production being glycolytic. Even if the iodo-acetate did not have its inhibiting effect because the of sea water is too high (Lunds-gaard, 1932), the absence of any effect with fluoride or hexose monophosphate seems to be conclusive. On the other hand, in an earlier paper Runnströom (1933 a) states that cytolytic treatments such as hypertonic and hypotonic sea water produce more acid in the presence of hexose monophosphate, which suggests that some form of glycolysis operates. This is confirmed by the experiments described in this paper.1
The situation as regards cytolysis can now be summarized. Runnström (1933 a) found that eggs treated with hyper- or hypotonic sea water (a treatment which frequently resulted in cytolysis) produced acid and the effect was enhanced by hexose monophosphate. Glycolysis therefore seems probable. Runnström (1935) also found that eggs cytolysed by freezing and thawing produce acid and the effect was not enhanced by hexose monophosphate. Nor was it inhibited by iodoacetate2 or fluoride. Glycolysis is precluded. Phlorizin markedly inhibits acid production in eggs cytolysing under the action of phlorizin. Glycolysis seems probable. Either Runnstrôm’ s results are inconsistent, or different forms of cytolysis produce different kinds of acid, hypertonic sea water being similar, in the type of acids it produces, to saponin but not to freezing and thawing.
SUMMARY
M/500 phlorizin in sea water decreases the acid production of cytolysed Echinus esculentus eggs. This suggests that the acid may be glycolytic in origin.
ACKNOWLEDGEMENTS
I wish to record my thanks to the Director and Staff of the Scottish Marine Biological Station, Millport, for the facilities I enjoyed while working there, and to Dr T. Mann, of the Molteno Institute of Parasitology, Cambridge, for his valuable criticisms of this paper.
REFERENCES
This paper is not concerned with carbohydrate breakdown without phosphorylation (Bumm & Fehrenbach, 1931).
This effect was firat noticed in Echinus esculentus eggs by Gray (unpublished), to whom I am indebted for suggesting a further investigation of the effect.
The radius of unfertilized E. esculentus eggs was 82.6 ± 4.2µ (S.E. of mean).
The whole of this analysis depends on the amount of saponin tipped in being sufficient completely to cytolyse all eggs in the manometer vessels. If this precaution is not taken the results become exceedingly difficult to analyse.
Tyler and Schultz’s (1932) experiments on the eggs of Urechis caupo and the more recent experiments of Tyler (1937) on various marine eggs may indicate that activation is partially reversible.
The results described in this paper have very recently been repeated using the more accurate Warburg differential method (1924).
Runnström (19336) showed that the cell membrane of Arbacia eggs is permeable to iodoacetate. Ellis (1933) also considers that iodo-acetate has no effect on fertilization or division, but penetrates the fertilized eggs, as evidenced by decreased glutathione content in fertilized eggs treated with iodo-acetate.