Iodine Compounds and Fertilisation: VII. The Nature of the Egg Secretions and the Theory of Fertilisation

At least the following three types of substance are known to occur in egg secretions, but it must not be supposed that all of them are necessarily present in the secretions of any one species.

• (1) The iso-agglutinin (Lillie, 1914). This substance causes a non-toxic, temporary and reversible agglutination of the sperm of the species from which it is derived. It is specific and is known to be colloidal (Lillie). It is perhaps an enzyme (Richards and Woodward, 1915). It was shown by Lillie that its presence in the egg is essential for the activation of the egg, and that it is identical with or at least very closely related to a substance which he named “fertilizin,” the “activation” of which, as a result of the entrance of the sperm, he believed to be an essential preliminary to the activation of the egg.

• (2) The hetero-agglutinin (Lillie, 1914). This causes a permanent, irreversible and usually toxic agglutination of foreign sperm. Its action on sperm of its own species is presumably masked by the action of the iso-agglutinin. It was shown by Lillie to be non-specific in its action, but not all types of foreign sperm are agglutinated by it. It was also shown by Lillie to be distinct from the iso-agglutinin. Its chemical nature is unknown. It is not present in the egg-water of all species (Sampson, 1922), nor is it always present in egg-water obtained from healthy eggs of species which sometimes produce it (Strongylocentrotus, Lillie, 1921).

• (3) A volatile substance which Clowes and Bachman (1921) found to be present in the secretions of Asterias and Echinarachnius. This substance could be distilled from the egg-water and was effective in activating the sperm of these species. They found that certain complex alcohols (propyl, amyl and cinnamyl alcohols) were also effective in activating the sperm and suggested that the substance which they distilled was of this nature. Such a substance might very probably be formed as a product of fermentation and might probably be present in egg-water, especially in artificially concentrated egg-water made in the usual way by aerating a large quantity of eggs in a small quantity of sea water. There is no evidence of its occurrence under natural conditions, but it is equally impossible to demonstrate the presence of any of the other components of the secretions except when the eggs are crowded, owing to the infinitesimal amounts secreted by the eggs.

To this list must, more doubtfully, be added the lipolysin, which Woodward (1918) claimed to have separated from the egg-water of Arbacia and Asterias, and which she and Glaser believed to be the cause of the autoparthenogenesis which Glaser observed (1914). Both the occurrence of autoparthenogenesis and the existence of the lipolysin have been denied by Just (1928, 1929). The existence of this substance must be considered doubtful until more evidence concerning it has been brought forward.

In the course of the series of papers of which this is one, thyroxine has been shown to produce similar effects to those of the secretions in :

• (1) The activation of the sperm and the prolongation of its active life (1930, 1931 a).

• (2) The hetero-agglutination of the sperm (1932 b).

• (3) The ripening of the egg (1932 a).

• (4) The prolongation of the fertilisable life of eggs exposed to a current of sea-water (1931 V).

• (5) The improvement of the development of over-ripe eggs (1932 a).

In considering these results, especially those concerning the fertilisable life of the egg, together with the results of Lillie’s work, the conclusion was reached (1932 a) that thyroxine is closely related in a chemical sense to the substance which Lillie called fertilizin, and which he showed to be very closely related to the isoagglutinin; and that conclusion has been confirmed by the fact that a substance having similar chemical properties to thyroxine and similar physiological action to the secretions can be extracted from the egg (1932 c).

So far little attempt has been made to bring these results and conclusions into harmony with the earlier work, except in so far as consideration of Lillie’s theory was necessary to reach the conclusions stated above. To attempt to bring all these facts into harmony, and so to approach a consistent theory which will take account of all the known facts concerning the chemical changes which occur at fertilisation is the object of the present paper.

The point at which the results given in these papers appear at first sight to be most clearly in disagreement with those of earlier workers concerns the two agglutinins. Lillie (1914) has shown very clearly that it is the iso-agglutinin which is essential for the activation of the egg, and not the hetero-agglutinin, which is not always present in the secretions. We have concluded that thyroxine is chemically related to fertilizin. Yet thyroxine resembles the hetero-agglutinin more closely than the iso-agglutinin in the type of agglutination which it causes and in its lack of specificity in causing agglutination (1932 b). Further, the iso-agglutinin is a very complex substance and perhaps an enzyme. It is undoubtedly far more complex than thyroxine. The chemical nature of the hetero-agglutinin is unknown, but its non-specific action suggests that it is simpler than the iso-agglutinin. All this evidence seems to show that thyroxine is related to the hetero-agglutinin more closely than to the iso-agglutinin, a conclusion which appears to be antagonistic to the belief that it is related to fertilizin, which is known to be very closely related to the iso-agglutinin.

It seems that these apparently contradictory facts can only be brought into harmony if it is supposed that the two agglutinins are themselves related. If we also suppose that the hetero-agglutinin is a much simpler substance than the iso-agglutinin and is one of the components from which the latter (or fertilizin) is built up in the cytoplasm of the egg, all the results fall into line¹. For then, accepting the close relationship of thyroxine to the hetero-agglutinin, we should expect that the iso-agglutinin could be formed from thyroxine as well as from the heteroagglutinin. And thus thyroxine might serve as a source of fertilizin to the egg, although it is most closely related to the hetero-agglutinin.

These assumptions also give a basis for the interpretation of the fact that foreign egg-water is able to improve the development of unripe and over-ripe eggs (1932 a). It is no great further assumption that the non-specific hetero-agglutinin can be built up in the eggs of species other than that from which it was derived into the iso-agglutinin of the species of the egg. It might therefore serve as a source of iso-agglutinin (or fertilizin) for the eggs of these species and so improve the condition of the eggs.

Further, the fact that the hetero-agglutinin is not always present in the secretions is to be expected on this view of the nature of the agglutinins. If the heteroagglutinin is a substance from which the iso-agglutinin is formed in the egg, it would presumably only remain in the uncombined form in the egg if it were originally present in excess, and would only be found in the secretions when this was so. It is therefore not surprising that egg-water formed from eggs of some females of a species might contain it and others not. This was what Lillie (1921) found to occur in Strongylocentrotus. On the other hand, the iso-agglutinin being necessary for the activation of the egg, must always be present in healthy eggs and in their secretions.

The results of Clowes and Bachman (1921) also seem at first sight to be in opposition to this theory of the egg secretions. The evidence presented in these papers has shown that thyroxine is able to cause both agglutination and activation of the sperm. If thyroxine is chemically related to the hetero-agglutinin, it is hard to avoid the conclusion that this substance is also able to produce both these effects. It has generally been thought, mainly on the evidence of Clowes and Bachman’s experiments, that the agglutinating and activating substances are distinct. It has been believed that the activating substance is the volatile substance which they distilled. The suggestion has already been made that a product of fermentation such as the volatile substance may be present in artificially concentrated egg-water and not in the secretions which diffuse from the egg under natural conditions. If, however, this substance were shown to be a normal component of the secretions, it is still not unlikely that the activation of the sperm is doubly secured by the presence of both this substance and the agglutinin in the secretions¹.

There is, however, another point in which their results appear to contradict the view of the secretions here proposed. They found (Clowes, personal communication) that the residue of the egg-water after the distillation was ineffective in causing activation of the sperm. It is unlikely that the heating of the water during the distillation would destroy a substance closely related to thyroxine. Thus, the hetero-agglutinin, if present in the egg-water, should have been present in the residue, and should have produced activation. But we know that the heteroagglutinin is not always present. It is true that it is normally present in Arbacia egg-water (Lillie, 1913), but Just (1919) failed to find it in that of Echinarachnius. There is no evidence at present that the iso-agglutinin can cause activation of the sperm, although it may seem probable that it can. If it were shown to be able to do so, the question whether the iso-agglutinin would be present in the residue after distillation would still remain. Lillie (1913) has shown that the iso-agglutinin of Arbacia is destroyed at 95° C. in 30-65 minutes and that of Nereis in 10–22 minutes. It therefore seems likely that the iso-agglutinin would be destroyed by the heating of the water during the distillation. And it is by no means certain that the destruction of the more complex iso-agglutinin would set free the simpler hetero-agglutinin.

If we accept the conclusion that thyroxine and the hetero-agglutinin are closely related, we must also conclude that the substance which was extracted from the egg is either very closely related to the hetero-agglutinin or identical with it. The facts that the method of extraction was one that leads to the isolation of thyroxine from other tissues and that this substance has similar physiological action to that of the secretions seem to necessitate this conclusion.

None of the evidence given in these papers bears upon the questions of the existence and nature of the lipolysin. Its presence in egg-water must still be considered a matter of doubt. The evidence is, however, in favour of Lillie’s theory that the iso-agglutinin is the component of the secretions which is essential to the activation of the egg, rather than that of Woodward (1918) that the lipolysin is the essential substance. For thyroxine causes agglutination and can also replace fertilizin.

This discussion has shown that none of the evidence is directly contradictory to the theory of the egg secretions which we have now reached. That theory may be shortly summarised as follows:

• (1) The hetero-agglutinin is closely related to thyroxine and to the substance which was extracted from the egg. It may be more complex than these substances but is simple as compared with the iso-agglutinin. It is at least one of the substances which causes the activation of the sperm.

• (2) The iso-agglutinin is a much more complex substance, perhaps an enzyme, and can be formed in the cytoplasm from the hetero-agglutinin, probably with other components. It is identical with or very closely related to fertilizin, and its presence is necessary for the activation and development of the egg. It is not known to cause the activation of the sperm, but it is not improbable that it may do so.

• (3) If the volatile substance of Clowes and Bachman is secreted from the egg under natural conditions, it appears to be a second means by which the activation of the sperm is secured.

• (4) The presence and nature of the lipolysin is considered doubtful.

• (5) There is no good evidence at present that either the volatile substance or the lipolysin is essentially related to the chemical changes of the activation of the egg.

This view of the nature of the secretions is a modification of the theory of Lillie (1914). It is not suggested that its truth is established by the evidence so far given. It is put forward rather as a working hypothesis which will probably need alteration in the light of further evidence.

Nor is it to be regarded as a complete theory of fertilisation. It is clearly not so. In an earlier paper (1924) the view was maintained that the first change in the egg after the sperm reaches its surface is a change in the conditions of the surface membrane, and that chemical changes in the cytoplasm succeed this first change. The weight of the evidence still seems to me to be in favour of such a view and none of the evidence given in these papers is opposed to it. We have here been concerned only with the nature of the substances which play a part in the (secondary) chemical changes which occur in the cytoplasm. Clear evidence that the changes of activation are more complex than those here discussed is given by the fact that fertilizin does not diffuse from the fertilised egg (Lillie), although it must still be present. Whether this is a result of the changed surface conditions, or, as Lillie believes (1914, etc.), of chemical changes in the fertilizin itself, or of both, it is not necessary to discuss here.

In one respect the theory here propounded would, if it were established, throw some light on the function of fertilizin in the egg. It is well known that there is a very great rise in the level of chemical activity of the egg at fertilisation, and thyroxine is a substance which is known to exert a controlling influence on the level of some forms of chemical activity in vertebrate tissues. If fertilizin is in some way related to thyroxine, it is difficult to avoid the conclusion that this substance is concerned in the manner in which the increased chemical activity of the egg is brought about, and that the necessity for its presence in the egg at fertilisation is associated with the necessity for this increase of chemical activity.

The results discussed in these papers have some bearings on the general physiology of iodine compounds. We have found that a substance resembling thyroxine plays a part in the chemical activity of cells so distantly related to vertebrate tissues as the eggs and sperm of Echinoderms. This seems to be evidence for the view that compounds of this nature are somehow concerned in the chemical activity of the animal cell in general, and this evidence is strengthened by the fact that it has been obtained from cells as primitive and as differently specialised as the egg and sperm. It is often thought that the negative results which have almost always been obtained from experiments in which invertebrate tissues have been treated with thyroxine make this conclusion improbable. Possibly the reason for these negative results is contained in the evidence collected in these papers. It has been shown that thyroxine has no demonstrable effect on the ripe egg and sperm, although its effects on both when they are unripe or over-ripe are definite. It may be suggested that it is not likely that any effect will be observed when healthy cells, functioning normally, are treated with a substance such as thyroxine which, on our view, they normally contain. Such a cell will contain sufficient of the substance for its needs and will not be affected when more of the substance is presented to it in the medium, unless the concentration is too great for the health of the cell, when the effect will be harmful. Most of the experiments in which invertebrate tissues have been treated with thyroxine are of this type, and the negative results obtained from these experiments give very little evidence that iodine compounds do not play as essential a part in the activity of the invertebrate as of the vertebrate cell. It is only when the amount of these substances present in the cell varies naturally, as it apparently does in the cells of the vertebrate body, or when the cells are not in a perfect condition of normal health, as in the experiments on unripe and over-ripe eggs and sperm, that positive results can be expected from treatment of the cells with these substances. In this respect such substances differ essentially from drugs which play no part in the normal activity of the cell.

Finally it is worth notice that the effects produced by treatment with thyroxine were immediate, both in the activation of the sperm and in the improvement of washed and over-ripe eggs. This is in contrast with the long latent period which is found in its action on the vertebrate. For instance, Gaddum (1930) found that the maximum increase in the basal metabolism of the rat usually occurred during the second day after administration of thyroxine. The results given in these papers seem to show that thyroxine produces its effects on the cell as soon as it reaches it. In spite of the fact that the latent period is as marked in the thyroidectomised as in the normal animal, and is therefore not the result of any action of the thyroid, we can only conclude that the latent period is in some way caused by the more complex conditions in the vertebrate body. At least, the evidence here given shows that this delay is not characteristic of the action of iodine compounds on the animal cell in general.

1. The results of the previous papers of this series are summarised and a theory of the nature of the egg secretions is suggested on the basis of these results and those of previous workers. It is suggested that the hetero-agglutinin is a substance closely related to thyroxine and the iso-agglutinin a more complex substance of which the hetero-agglutinin is an essential component.

2. The bearings of these results on the general physiology of iodine compounds are discussed.