Iodine Compounds and Fertilisation: III. The Fertilisable Life of the Eggs of Echinus Esculentus and Echinus Miliaris

Lillie (1914) has shown that eggs become unfertilisable if they are washed for a long time in a constantly changing current of sea-water, and he reached the conclusion that this was due to the loss of the secretions, which diffuse from the surface of the egg. At least one component of the secretions must remain within the egg, if activation is to take place. If this essential component of the secretions is also the component which is responsible for the similarity between the actions of the secretions and thyroxine upon the sperm, it is possible that thyroxine may be able to replace it, at least functionally, in the cytoplasm. If this is so, and thyroxine is allowed to diffuse through the surface of eggs which are losing their secretions in a current of sea-water, the fertilisable life of the eggs should be longer than that of eggs into which thyroxine was not diffusing as the secretions were being lost. Thyroxine will presumably diffuse into the eggs, if it is present in the medium, and its presence in the medium should therefore, if this hypothesis is true, prolong the fertilisable life of the eggs.

The following experiments were arranged with the object of testing the power of thyroxine to replace the secretions in this way. Samples of eggs from the same urchin were washed under similar conditions in currents of sea-water with and without thyroxine, and the length of the fertilisable life of the eggs in the different samples was compared. In addition to thyroxine, the several related substances which were used in the experiments discussed in the previous paper (1931)¹, were also used in these experiments, in order that any effects produced by them might be compared with any produced by thyroxine.

The objection may, perhaps, be raised to these experiments as providing an answer to the questions which they were intended to decide, that any prolongation of the fertilisable life produced by the presence of thyroxine might be due, not to diffusion of thyroxine through the surface of the egg, but rather to a decrease in the permeability of the surface layer produced by the presence of thyroxine outside the surface. By this means the loss of the secretions would be retarded, and the fertilisable life of the eggs consequently prolonged. But it seems very improbable that the permeability of the surface should be decreased by the presence of thyroxine in very dilute solution in the medium. There is no evidence in favour of such a possibility, and, further, the best known physiological action of thyroxine is its power to promote certain types of chemical change in the mammalian body, especially those of carbohydrate metabolism (cf. Kendall, 1929, pp. 134 ff.). It would seem unlikely that a substance which promotes chemical activity in the body, also reduces the permeability of the surface of the cell. If the presence of thyroxine in the surrounding medium results in a prolongation of the fertilisable life of the egg, it seems that the only probable conclusion is that it does so by diffusing into the egg and by action within the egg similar to that of the secretions.

In carrying out these experiments it was found that the method of removing the secretions by adsorption on charcoal (developed by Glaser, 1921) was inapplicable. It was found that charcoal adsorbed thyroxine and probably the other chemical substances. The only available method was therefore that which Lillie originally used, namely to remove the secretions by washing the eggs in constantly changing sea-water. Experiments of this type necessarily last many hours and to have passed a current of water, consisting of a solution of the substance, once over the eggs for this time would have required a very large amount of the solution. A more economical arrangement was devised by which a considerable quantity of the solution (650 c.c.) was kept circulating over a small number of eggs by means of a jet of air (see Fig. 1). So long as very few eggs were used, this arrangement was almost as efficient in washing away the secretions as a current passed only once over the eggs.

In the following tables the results of two experiments, one with the eggs of E. esculentus and one with those of E. miliaris, are given. In each experiment all the batches of eggs were taken from the same urchin. Samples were taken out of the apparatus at the stated intervals and fertilised in sea-water. The development of these samples was watched and recorded. These experiments are examples of several of the same type, all of which gave similar results.

All membranes which were visibly separated from the surface of the egg were included in the percentages given in the table.

Throughout each of these experiments the hydrogen-ion concentrations of the circulating solutions were kept identical with that of sea-water. The other conditions were kept as nearly as possible the same in the different solutions.

The length of life of the eggs in sea-water under the conditions of the experiments varied greatly in the different experiments. In some of the experiments the eggs were fertilisable after circulation for 12 hours in sea-water (Table II), and others gave no membranes and few divisions after circulation for $212$ hours. These variations occurred in the eggs of both species and were apparently associated with the condition (ripeness, etc.) of the urchin at the time at which the eggs were taken from it.

Although the results given in the tables are slightly irregular (probably mainly on account of diurnal variations of temperature and other conditions), it will be seen that the presence of thyroxine results in a very marked prolongation of the fertilisable life of the egg. This prolongation is seen both in the percentage of eggs which develop and in the regularity of the membranes and divisions. The action of thyroxine was perhaps more marked in its effect upon membrane formation than upon the subsequent divisions. This is probably because eggs, which do not form regular membranes at fertilisation, are able to recuperate to some extent later.

The results of this series of experiments also showed that the improved fertilisability due to the presence of thyroxine resulted in better development in later stages, at least as far as the blastula stage.

In Table III the results of two experiments are given in which the fertilisable life of eggs in a current of sea-water containing thyroxine is compared with that of eggs aerated in a small quantity of sea-water, as well as with that of eggs in a current of sea-water alone. Lillie (1914) has shown that eggs under aeration in a comparatively small quantity of sea-water remain fertilisable for a much longer time than those exposed to a current. He ascribed this result to slower diffusion of the secretions from eggs in a small quantity of water, which soon becomes saturated with the secretions. The object of these experiments was, therefore, to compare the extent of the prolongation of the fertilisable life produced by the presence of thyroxine with that produced by the retention of the secretions within the egg.

These experiments are examples of several of this type, all of which gave similar results.

In t-he first of these experiments the eggs under circulation in the presence of thyroxine were fertilisable for a considerably longer time than those under aeration. This was not so in the second of the experiments and was almost certainly due to the less favourable conditions (in the supply of oxygen and removal of carbon dioxide, etc.) of the eggs under aeration. In order that the water surrounding these eggs may become saturated with the secretions, it is necessary that the eggs should be somewhat crowded, and crowding of the eggs necessarily leads to unhealthy conditions.

The results of these experiments show that the prolongation of fertilisable life produced by the presence of thyroxine is at least as long as that produced by the slower loss of the secretions in the eggs under aeration. From the results of several experiments, in which the eggs under aeration lived almost as long as those in a current of water containing thyroxine, and in view of the less healthy conditions of the eggs under aeration, it seems that there is no reason to suppose that the presence of thyroxine produces any greater prolongation of life than the retention of the secretions within the egg. Both treatments prolong the life of the eggs to about the same extent.

Results of experiments with some chemical substances related to thyroxine have been given in these tables. Experiments were carried out with the whole series of substances mentioned above (p. 194), and with changes in the hydrogen-ion concentration of the circulating sea-water. Their results may be summarised as follows :

1. Des-iodo-thyroxine produced a prolongation of the fertilisable life which was sometimes considerable (Table I) and sometimes much less so (Table II). Its effect was usually greater in experiments in which the eggs of E. esculentus were used.

2. Iodine (dissolved in a solution of potassium iodide of two and a half times the strength of the iodine) produced a slight prolongation of the life of eggs at a concentration of 1/1,000,000 and 1/2,000,000. At greater concentrations it was toxic. Its effect was usually greater in experiments in which the eggs of E. miliaris were used.

3. Di-iodo-tyrosine (Table I) and all the other chemical substances were ineffective at a concentration of 1/50,000.

4. Variations in the hydrogen-ion concentration of the sea-water between pH 7·2 and pH 9·4 had only slight effects upon the length of the fertilisable life. The addition of acid to the sea-water produced a slight and irregular lengthening of the fertilisable life. In abnormally alkaline sea-water there was an equally slight shortening of the fertilisable life.

All the positive effects in these experiments were without exception much less than the effect produced by thyroxine. Des-iodo-thyroxine produced a more definite effect than any other of these substances.

The experiments recorded in this paper show :

1. That the presence of thyroxine in the sea-water surrounding eggs which are losing their secretions prolongs the fertilisable life of the eggs.

2. That the prolongation of fertilisable life so produced is of approximately the same extent as that produced by the retention of the secretions within the eggs.

3. That des-iodo-thyroxine has a slight effect in prolonging the life of eggs, but that no other of several substances chemically related to thyroxine has this effect.

4. That alterations of the hydrogen-ion concentration of the sea-water in which eggs are being washed (between pH 7·2 and pH 9·4) do not produce any prolongation of fertilisable life comparable with that produced by thyroxine.

Reasons have been given for believing that the effect produced by thyroxine is not due to a reduction of the permeability of the surface layer of the eggs, but rather to diffusion of thyroxine into the egg and to some effect produced by it within the surface of the egg.

If these conclusions are accepted, it seems that the following further conclusions must also be accepted :

1. The unfertilisable condition of eggs which have lost their secretions is due, at least in the main, to the loss of a component of the secretions which can be functionally replaced by thyroxine. Unless more than one component can be so replaced, it must be due to the loss of one component only.

2. Since thyroxine and the secretions have been shown in the previous paper to produce the same effects upon the oxygen consumption of the sperm, it must be concluded that the component of the secretions essential for the activation of the egg is also that which produces these effects upon the sperm.

3. Since thyroxine is known to raise the level of certain types of chemical activity in the mammalian body, and since there is a very large rise in the level of chemical activity in the egg at fertilisation, the conclusion is at least probable that the necessity for the presence of this component of the secretions in the egg at fertilisation is due to some part, similar to the known physiological action of thyroxine, which this component of the secretions plays in the chemical changes of activation.

It remains to consider whether these results allow us to retain the conception that this component of the secretions is similar to thyroxine only in its physiological activity, or whether they compel the further conclusion that these bodies are related in chemical structure. No definite answer is given to this question by the results here recorded. All the three phenomena so far discussed in these papers (the immediate activation of the sperm of E. esculentus, the prolongation of the active life of that of E. miliaris, and the prolongation of the fertilisable life of the eggs of both species) might, perhaps, be caused by the introduction of a substance which increased the maximum rate of chemical processes in the protoplasm of the cells, and possibly by the introduction of any substance which acted in this way. But the variety of these effects, and in each phenomenon the close similarity (or more truly identity) between the effects produced by the secretions and thyroxine, make it very improbable that a chemically unrelated body could so accurately reproduce the action of the secretions in all of them. This seems even more improbable when it is remembered that the egg and sperm are both extremely complex but very different chemical systems.

It might perhaps be thought that the varied means by which parthenogenesis can be initiated in the egg show that unnatural means may take the place of the natural in some at least of the phenomena of activation, and that there is therefore no reason why this should not also be true of the changes which result in the increase in chemical activity in the egg at fertilisation. But the initiation of parthenogenesis is probably due primarily to the production of a change in the surface layer of the egg, which allows the changes of chemical activation to occur later in the cytoplasm of the egg itself (cf. Carter, 1924). These latter changes are probably not directly produced by the parthenogenetic agent. It is far more likely that unnatural means could replace the natural in producing a change of the surface layer of the egg than in the complicated changes of activation in the cytoplasm. It is still less likely that the replacement in these latter changes could be so complete as to lead to normal development of the larva. It has been shown in the experiments recorded in this paper that this is true of the replacement of the secretions by thyroxine within the egg, at least as far as development to the stage of the blastula is concerned. Thus, although these experiments give no proof that thyroxine is chemically related to a component of the egg-secretions, they make it very probable that this is so.

1. The presence of thyroxine in a current of sea-water in which eggs of either E. esculentus or E. miliaris are being washed prolongs the fertilisable life of the eggs.

2. Under these conditions the fertilisable life of the eggs is of approximately the same length as that of eggs which are aerated in a small quantity of sea-water.

3. Des-iodo-thyroxine and free iodine also prolong the life of eggs, when they are present in the medium, but the prolongation produced by them is much less than that produced by thyroxine. No other of several chemical substances related to thyroxine produce this effect. Changes in the hydrogen-ion concentration of the medium between pH 7·2 and 9·4 have very little effect upon the length of life of the eggs.

4. It is concluded that the component of the secretions which is essential to the activation of the egg can be replaced by thyroxine, and is therefore the same as the component which produces effects similar to those of thyroxine on the oxygen consumption of the sperm. It is also concluded that it is probable that this substance, besides being similar to thyroxine in physiological action, is related to it in chemical structure.

The expenses of this research were defrayed, in part, by a grant from the Government Grant Committee of the Royal Society.