Iodine Compounds and Fertilisation: II. The Oxygen Consumption of Suspensions of Sperm of Echinus Esculentus and Echinus Miliaris

In all these experiments the oxygen consumption of the suspensions was measured in Barcroft manometers, and the results are therefore not direct estimations of the activity of the suspensions. The primary object of the experiments was not to estimate the activity of the suspensions, but to compare the effects upon their oxygen consumption, which Gray (1927) has shown to be produced by the presence of egg-secretions, with any produced by thyroxine. It is therefore not essential to the argument to decide whether measurement of the oxygen consumption is equivalent to measurement of the activity of the suspension. However, it is prima facie probable that this is true, and there is no evidence that it is not. In this paper, where the point arises, it will be assumed that measurement of the oxygen consumption also gives a measurement of the activity of the suspension.

The experiments upon the sperm of E. esculentus were performed with respirometer bottles fitted with a graduated side tube. By means of this side tube a drop of a concentrated sperm suspension can be introduced without removing the bottle from the instrument, and thus slight temperature changes during the addition of the sperm are avoided. This type of bottle has a further advantage. If the instrument is kept closed when the drop is introduced, the alteration of the position of the liquid in the manometer can be used as a very accurate estimate of the size of the drop. The movement of the liquid must, however, be previously calibrated for drops of different size. If the amount of sperm is estimated in this way, the chief error which remains is probably that caused by differences in the concentration of the sperm in the drop, but these were found to be small when samples of the same suspension were used in all the instruments.

For reasons to be given below (p. 186) it was found that the quantity of sperm used in these experiments must be less than one drop of dry¹ sperm to the 5 c.c. of liquid in the bottle. The suspension in the side tube was therefore diluted to three times the volume of the dry sperm before being placed in the instrument. It is necessary to leave this diluted suspension in the side tube for 15–20 minutes, while the instrument is becoming adjusted to the temperature of the bath in which it is being shaken, but at this slight dilution the sperm of E. esculentus is inactive except near a surface exposed to the air, and its behaviour after it was added to the liquid was found to be the same as when undiluted sperm was used.

Owing to the smaller size of E. miliaris, and the small quantity of sperm which can be obtained from it, it would have been necessary to dilute the sperm more, if the side tubes of these bottles had been used. The effect of greater dilution on the sperm was found to be irregular. For this reason the usual type of bottle without a side tube was used for experiments on the sperm of this species. The sperm was added with a pipette after the bottle had been opened, care being taken to reduce temperature changes in the bottle during the operation to a minimum. In spite of all possible precautions, the readings of the manometer were usually irregular during the first 5 minutes of the experiment, and these readings have been neglected in drawing the curves relating to the experiments on the sperm of this species.

In each experiment, samples of the same concentrated suspension of sperm were used in all the instruments. The volume of the drop added to each instrument therefore gave an accurate estimate of the amount of sperm present. In spite of this, estimations of the total nitrogen content of the concentrated suspension were often made by the micro-Kjeldahl method. When only one experiment is being considered, these serve no other purpose than to define approximately the concentration of the suspension. But it was often necessary to compare the results of two or more experiments, and it will be found that in some of the figures more than three curves are drawn. A set of three similar manometers were used, and therefore, in these figures, the results of more than one experiment are given. It was found, for reasons given below (p. 180), that the same concentrated suspension of sperm could not be used in two successive experiments. Further, if sperm from different urchins was used, the oxygen consumption of suspensions containing the same N₂ content varied greatly. As the content of N₂ per c.c. was the only means of comparing the results of different experiments, no satisfactory comparison could be made in a single figure between experiments on sperm from different urchins. However, it was possible to perform successive experiments on sperm taken from different gonads of the same urchin (see p. 181), and, when this was done, the oxygen consumption per mg. N₂ was found to be comparable. Whenever the results of two experiments are compared, the sperm was always obtained in this way, and the results are given as the consumption per mg. N₂ present in the suspension.

In the experiments with the sperm of E. esculentus, the curves have been corrected in scale for variations in the size of the drop from 0·035 c.c., which was its average size. Since the sperm of E. miliaris was added directly with a pipette, it was impossible to apply this correction to experiments on the sperm of this species, but it was found that the size of the drop added with the pipette varied less than that added from the side tube.

It will be clear from the results of the experiments given below (pp. 182, 187) that it was necessary, whenever the consumption of suspensions in different media were to be compared, that the hydrogen-ion concentrations of the media should be controlled as accurately as possible. The hydrogen-ion concentrations were always carefully adjusted before the experiment, and are given as those at the time at which the experiment started, i.e. after the carbon dioxide had been removed from the medium by the shaking of the instrument, and absorbed by the drop of strong NaOH in the cup at the top of the bottle. The values at this time were obtained by adding indicators to the media in the bottles on the side of the instrument to which sperm was not to be added. The sperm releases carbon dioxide in amounts varying with its activity, and the hydrogen-ion concentration therefore varies during the experiment with the activity of the sperm. No definite value, which will be true throughout an experiment, can be given for this reason. But, when very dilute suspensions are used, as in all the experiments discussed in this paper, these variations are not great. A suspension of the sperm of these species, at the concentration used in these experiments, in sea-water (normally at pH 8·3 and at pH 8·6 after shaking in the instrument) is at pH 8·4−8·5, while the sperm is maximally active.

In many of the experiments on the sperm of E. esculentus the sperm was fully active at the end of the experiment. There was approximately the same amount of sperm in each instrument, and the hydrogen-ion concentration of the suspensions in the different instruments should therefore have been identical at the end of the experiment, since they were so at the beginning. The accuracy of the control of the hydrogen-ion concentration could, therefore, be checked by estimating that of the suspension at the end of the experiment. This was done by adding indicators to the suspension immediately after the experiment. This method gave only rough estimations of the absolute hydrogen-ion concentration, but it allowed even small differences between the suspensions to be recognised. The results of these estimations are given in the descriptions of the figures.

With the object of forming some estimate of the specificity of the action of thyroxine, its effects have been compared in these experiments with those of several related substances. Des-iodo-thyroxine, tyramine, di-iodo-tyrosine, tyrosine, tryptophane, potassium iodide, potassium iodate and free iodine were chosen for this purpose. The action of adrenaline was also observed. These substances were obtained from the British Drug Houses. The thyroxine used was the synthetic substance and was of the more active, laevo-rotatory form.

In all the experiments it was necessary to make up solutions of known strength of the various chemical substances in sea-water. Solutions of thyroxine, des-iodo-thyroxine, tyramine, di-iodo-tyrosine, tyrosine, and tryptophane were made by dissolving the substance in N/10 sodium hydroxide so as to form a 1 per cent, solution by weight. This solution was then added in the correct proportion to sea-water, and the hydrogen-ion concentration of the final solution adjusted to that of sea-water with N/10 hydrochloric acid. The concentration of the substance in the final solution was never greater than 1/50,000, and the decrease of osmotic pressure caused by the addition of the concentrated solution and of the acid was therefore never more than 1/250 of that of sea-water. In a few experiments the effect of this change in osmotic pressure was controlled by observing the behaviour of the sperm in sea-water to which a corresponding proportion of distilled water had been added. The behaviour was always found to be unaltered by the dilution.

With the object of avoiding any errors due to possible breakdown of the chemical substances in solution, fresh concentrated solutions of the substances were always made up for each experiment.

When a solution of thyroxine in sea-water is made up in this way, a slight white precipitate is formed. The nature of this precipitate is unknown. It occurs when much lower concentrations than 1/50,000 of the drug are added to sea-water. It remains after the neutralisation of the alkali, and is therefore not caused by precipitation of salts from the sea-water. It consists of crystals which are mostly different in form from those of thyroxine, and the greater part of it is therefore not thyroxine itself. Probably, it is in the main some salt of thyroxine, but it may consist in the more concentrated solutions partly of undissolved thyroxine. Whatever the nature of this precipitate, it seems to be related to thyroxine, and its occurrence makes it probable that the solution of thyroxine in sea-water finally obtained is very much weaker than would correspond to the amount of the drug added. The concentrations of thyroxine given in this (and in the previous) paper refer to the amount added in solution in sodium hydroxide, and they must not be taken as referring to the actual concentration of the drug in the sea-water.

A similar precipitate does not occur when any of the other substances mentioned above are dissolved in sea-water at a concentration of 1/50,000.

Adrenaline was dissolved in N/10 hydrochloric acid, and the hydrogen-ion concentration of the final solution adjusted with N/10 sodium hydroxide. The drug breaks down rapidly in sea-water forming a pink solution. The colour is recognisable after 10 to 15 minutes at room temperature, but it continues to darken for at least 1 to 2 hours.

Potassium iodide and potassium iodate were made up in i per cent, solution in sea-water and diluted to the required strength. Iodine was used either in solutions formed by saturating sea-water by aerating it strongly for some hours in the presence of solid iodine, and diluting the solution to the required strength, or by dissolving known quantities of iodine with two and a half times the quantity of potassium iodide.

If an urchin appears from the condition of its gonads to be perfectly mature, it will often shed its sperm spontaneously after it has been opened, or slight pressure on the gonads will cause it to do so. Sperm obtained in either of these ways from a mature urchin usually activates at once in sea-water, and its oxygen consumption remains constant at the initial rate for almost an hour. On the other hand, if sperm is taken from the testis directly (by removing it from the animal and cutting it up in a watch-glass), the oxygen consumption is often at first very low but increases in 20 to 40 minutes to a rate which remains constant and is identical with that of a suspension of sperm shed at the genital pores containing the same quantity of N₂ per c.c. These differences in behaviour are shown in curves I and II of Fig. 1. The sperm from the genital openings of this urchin did not activate to the full extent immediately after the start of the experiment, but it will be seen that the initial consumption of this sperm was much higher than that of the sperm taken from the testis. In other experiments sperm shed at the genital pores often activated without any delay (Fig. 3, curve 1).

There can be no doubt that these differences in the behaviour of the sperm are caused by differences in its ripeness¹. An urchin which is clearly immature never gives sperm which activates at once, however it is obtained. It was found that sperm from a few urchins, which appeared to be perfectly mature, activated immediately, even when it had been taken from the testis.

If a sample of dry sperm, which was not ripe when it was taken from the urchin, is allowed to stand for some hours, it ripens gradually. An example of this is given in curve III of Fig. 1, which shows the oxygen consumption of another sample of the same sperm as that from which curve II was derived, after an interval of 3 hours.

Very unripe sperm may fail to ripen after it has been taken from the urchin.

Sperm does not ripen if it is left in undamaged gonads of an urchin which has been opened, and the gonads are covered with sea-water.

The fact that dry sperm ripens after it has been taken from the urchin made it impossible to use the same sample of dry sperm for two successive experiments, if the experiments are concerned, as are all those to be discussed in this section, with the increase in the oxygen consumption of unripe sperm after the beginning of the experiment. By preserving the dry sperm in undamaged gonads two or more experiments may be performed with the sperm of the same urchin, and it was found that the oxygen consumption of the sperm per mg. N₂ was then comparable (see p. 178 above). However, the ripeness of the sperm in the different gonads sometimes varies, and it is therefore necessary to test the ripeness in each experiment by the behaviour of the sperm in sea-water. This was always done, when the sperm of the same urchin was used for more than one experiment, and where, in the following figures, more than three curves are drawn, and the figure therefore gives the results of two experiments, the oxygen consumption of the sperm in sea-water was identical in the two experiments.

If a sample of dry sperm is kept for some hours after it has become ripe, it becomes over-ripe and ages gradually. During this process, it still activates at once on dilution to a rate of oxygen consumption which remains constant for at least an hour, but this rate falls continuously as the sperm ages.

In Figs. 2 and 3 curves are given showing the oxygen consumption of similar suspensions of sperm in sea-water to which different amounts of sodium hydroxide and hydrochloric acid had been added.

It will be seen that increased alkalinity of the medium above a certain value causes unripe sperm to activate immediately, but that the oxygen consumption decreases in these media more rapidly than in sea-water, and more rapidly the more alkaline the medium. There appears to be a hydrogen-ion concentration in the neighbourhood of pH 8·8−8·9, at which the immediate activation first takes place in the sperm used in this experiment. The facts previously stated show that this value varies with the ripeness of the sperm.

On the other hand, the activation of the sperm is delayed in media less alkaline than sea-water (Fig. 3). Sperm which activates immediately in sea-water does so slowly when the pH is 7·4.

Thus it appears that, with regard to its activation on dilution, ripening of the sperm consists of a movement in the acid direction of the least alkaline hydrogen-ion concentration at which the sperm activates immediately. Unripe sperm will only activate immediately in abnormally alkaline sea-water, ripe sperm does so in normal sea-water.

Unripe sperm can be observed under the microscope to activate much more rapidly both in egg-water and in sea-water containing thyroxine than in sea-water. No effect upon ripe sperm can be seen. The experiments on the oxygen consumption of the suspension gave results which were in agreement with these observations. An example of these results is given in Fig. 4. It will be seen that both egg-water and thyroxine produce immediate activation of the somewhat unripe sperm which was used in this experiment, and that the oxygen consumption of the suspension is identical in these two media. Other experiments showed that neither egg-water nor thyroxine in this concentration had any effect upon the oxygen consumption of ripe sperm.

In Figs, 1 and 3 other curves showing immediate activation in sea-water containing thyroxine have been given. It was shown in Fig. 3 that thyroxine produces this immediate activation at pH 7·4, although even ripe sperm does not activate immediately in sea-water of this acidity. There is thus a parallel between the effects of thyroxine and those of alterations in the hydrogen-ion concentration of the medium.

If a much denser suspension of unripe sperm is used, the oxygen consumption is unaltered by the presence of thyroxine. In Fig. 5 the results of two experiments in which one, three and five drops of a concentrated suspension were added to 5 c.c. of a solution of thyroxine in sea-water are given. The concentrated suspension was of the strength usually used in these experiments (three times diluted dry sperm) and contained 0·14 mg. of nitrogen per drop (0·035 c.c.). The full effect of the presence of thyroxine was obtained when one drop was added, but considerably less than the full effect when three drops were added, and very little effect when five drops were added. It appears that the activation is not produced unless the spermatozoon receives a sufficient dose of thyroxine, and that in the denser of these suspensions not sufficient thyroxine was present. The amount of thyroxine added to the suspensions in this experiment was 0·1 mg. in each case, and this produced the full effect in sperm containing 0·14 mg. N₂ but not in sperm containing 0·42 mg. But it must be remembered that by far the larger proportion of the thyroxine was precipitated, and, therefore, probably, largely ineffective. No definite estimate of the amount needed per mg. N₂ to produce activation can be given.

This result showed that very dilute suspensions must be used in all experiments in which the effects produced by thyroxine were to be observed. It was for this reason that a suspension obtained by adding one drop of dry sperm, diluted to three times its volume, to 5 c.c. of sea-water was used in all the experiments on the effect of thyroxine and other drugs. This density was chosen as one at which thyroxine produces its full effect, and one which would give sufficiently large readings on the manometer. Since it was desirable for purposes of comparison that all the experiments should be as similar as possible, this strength was always used.

The rate of oxygen consumption of over-ripe sperm is increased to some extent, but not to the original level, by the presence of the secretions and of thyroxine.

The power of producing immediate activation in unripe sperm of this species is not confined to thyroxine and the egg-secretions. Several of the series of chemical substances, mentioned above, were found to produce the effect, but the results of experiments in which they were used were somewhat irregular. In most experiments it was found that des-iodo-thyroxine, tyramine and di-iodo-tyrosine (1/50,000) produced the full effect in suspensions of the same density as those on which thyroxine was effective, but in some experiments only a partial effect was produced by these substances¹. Iodine (at the greatest concentration which did not kill the sperm) and inorganic iodine compounds (1/50,000) were ineffective. Adrenaline was toxic at concentrations of 1/50,000 and 1/60,000, and ineffective at lower concentrations. Tyrosine and tryptophane sometimes produced a partial effect and were sometimes entirely ineffective. The effect of these drugs apparently varies with the condition of the sperm, some samples being able to make use of substances which other samples cannot use. It is clear, however, that thyroxine produces a more definite and regular effect than any of these substances.

When it activates at all on dilution in sea-water, the sperm of E. miliaris, in all normal circumstances, activates to its maximum level of activity at once. No question of any effect of egg-water or chemical substances in hastening its activation therefore arises. Egg-water has however a different effect upon this sperm. Gray (1927) has shown that there is frequently a falling off in the activity of a suspension of the sperm to a value of about 40 per cent, of its initial activity in the first hour of its active life. This decrease of the activity of the sperm does not occur in egg-water, and it is with it that we are concerned in this section.

The decrease of activity is, however, very variable. It is very marked in some samples of sperm (e.g. Fig. 8, curve II), and in others it is unrecognisable (Fig. 6, curve II). These variations, like those in the rate of activation of the sperm of E. esculentus, seem to be due to differences in the ripeness of the sperm. If the gonads of the urchin from which the sperm is derived appear to be perfectly mature, and especially if the experiment is performed in the middle of the breeding season, the sperm gives a constant rate of consumption during the first hour. The less mature the urchin is, the greater is the decrease in the activity of the sperm.

The oxygen consumption of the sperm of this species is as markedly altered by changes in the hydrogen-ion concentration of the medium as is that of E. esculentus, but the alterations are different in kind. In Fig. 6 curves are given showing the oxygen consumption of samples of the same sperm at hydrogen-ion concentrations on the alkaline side of that of sea-water. It will be seen that sperm that gives a constant rate of consumption in sea-water gives a marked decrease of the consumption at pH 9·0 and that the decrease is still more marked at pH 9·3. A rise in the alkalinity of the medium emphasises the decrease of activity, and produces it when it is not present in sea-water.

Changes in the acid direction produce the opposite effect. Sperm, which gives a marked decrease in activity in sea-water, gives a constant rate of consumption in less alkaline media (Fig. 7).

It was mentioned above (p. 182) that the sperm of E. esculentus also shows a decrease in activity in alkaline media. It seems that this decrease in the activity of the sperm is the same phenomenon in the two species. That this phenomenon is different in nature from the activation of the sperm of E. esculentus in abnormally alkaline sea-water is shown by the fact that the effect of changes in the hydrogen-ion concentration is in the opposite sense in the two phenomena. The effect of alkalinity is to hasten the activation of the sperm of E. esculentus, and therefore to increase its activity at the beginning of the experiment. It produces a decrease of activity (with time) in the sperm of E. miliaris.

If we are right in believing that the presence or absence of this decrease in the activity of the sperm of E. miliaris in sea-water is the result of variations in the ripeness of the sperm, it seems that ripening, so far as this phenomenon is concerned, consists in an alteration of the behaviour of the sperm towards the hydrogen-ion concentration of the medium, as we found it did in considering the activation of the sperm of E. esculentus. But, again, the alteration is in the opposite sense. As it ripens, the sperm of E. miliaris gives a constant consumption in more and more alkaline media, and thus the most alkaline medium, at which it gives the constant consumption, moves in the alkaline direction. Ripe sperm of E. esculentus activates immediately in sea-water, less ripe only in abnormally alkaline sea-water. As it ripens, it activates in less and less alkaline media.

In the previous paper (Carter, 1930) it was shown that thyroxine resembles the egg-secretions in prolonging the active life of the sperm of this species, but the prolongation produced by thyroxine in the experiments discussed in that paper was by no means so great as that which the egg-secretions produced. The experiments on the sperm of E. esculentus, discussed in the previous section, showed that it was necessary to use a very dilute suspension of sperm, if the full effect of thyroxine was to be obtained, and much more dilute suspensions than those used in the experi-’ ments discussed in the previous paper. These experiments were therefore repeated with suspensions of the same density as those used in the experiments on the sperm of E. esculentus, namely, one drop of three times diluted dry sperm to 5 c.c. of liquid.

This work was undertaken in the middle of the breeding season and the urchins were almost all ripe. Their sperm very frequently showed hardly any decrease of activity during the first hour after dilution in sea-water, and experiments on the action of egg-secretions and thyroxine in reducing this decrease therefore failed when sea-water was used as the medium. However, the results discussed on the previous page had shown that even ripe sperm gives a marked decrease in alkaline sea-water. In these circumstances most of the experiments were carried out at pH 9 0 (as estimated after shaking). The few experiments which were done with unripe sperm in sea-water at the normal hydrogen-ion concentration gave exactly similar results. The conclusions drawn from these results may therefore be taken as true in general of the action of these substances on the decrease of the activity of the sperm, and not only of their action in abnormally alkaline sea-water.

It was found that the decrease in the activity of the sperm did not occur in sea-water at pH 9·0, when either egg-secretions or thyroxine were present. When either was present, the suspension gave a constant consumption for at least one hour and the consumption was identical in the presence of these two substances. It was also often identical with that of a similar suspension in sea-water at pH 8·6 without thyroxine or egg-secretions. Thus the effects of the presence of thyroxine are similar to those of changes in the hydrogen-ion concentration in this phenomenon as in the others previously mentioned.

Examples of these results are given in Figs. 8 and 9. The effect of thyroxine is also shown in Fig. 6, curve I, and in Fig. 10, curve I.

These experiments, therefore, showed that the failure to obtain complete similarity between the effects of thyroxine and egg-secretions in the experiments reported in the previous paper was caused by the use of too dense suspensions in those experiments.

If the alkalinity of the medium is increased beyond a hydrogen-ion concentration of approximately pH 9·4, the presence of thyroxine has no effect upon the activity of the sperm. This is shown in the curves of Fig. 6. Curve I gives the oxygen consumption of the sperm in the presence of thyroxine at pH 9·2. In sea-water without thyroxine, but of the same alkalinity, the consumption is that shown in curve IV. The difference between these two curves gives the effect produced by thyroxine at this hydrogen-ion concentration. Curve V gives the consumption in the presence or thyroxine at pH 9·6. It is lower than the consumption in sea-water at pH 9·3 and its form shows that the effect of thyroxine is not produced at this hydrogen-ion concentration.

A series of experiments were carried out on the effects produced by other chemical substances on the decrease in the activity of the sperm of this species, but the results were even more inconclusive than those of the similar experiments on the activation of the sperm of E. esculentus. It appeared that the effect was more specific to thyroxine than the effect on the activation of the sperm. None of the other substances used were found to give the full effect. Owing to the great sensitivity of this phenomenon to changes in the hydrogen-ion concentration of the medium, it was very difficult to decide whether a partial effect was caused by small changes of that kind or by the presence of the substance. Fairly good evidence was obtained that des-iodo-thyroxine at a concentration of 1/50,000 caused some prolongation of the active life of the sperm (Fig. 10), and some evidence that tyramine and di-iodo-tyrosine also did so.

The results of the experiments discussed in this paper may be summarised as follows :

1. Spermatozoa of E. esculentus.

   • (a) The rate of activation of samples of the sperm of this species after dilution in sea-water varies greatly.

   • (b) This variation is associated with the changes occurring in the sperm as it approaches the condition in which it is normally shed. For this condition the term “ripe” has been used in this paper. As it becomes ripe, the sperm activates more quickly ; fully ripe sperm activates at once.

   • (c) Activation is greatly accelerated by the presence of the egg-secretions (Gray, 1927).

   • (d) The presence of thyroxine produces an effect exactly similar to that of the secretions.

   • (e) Some substances related to thyroxine produce this effect in some samples of sperm, either partially or completely.

2. Spermatozoa of E. miliaris.

   • (a) The rate of decrease in the activity of the sperm of this species in the first hour after dilution is also very variable.

   • (b) It is less at the height of the breeding season, and in general appears to be less in the sperm of more mature urchins. Its amount is probably associated with the ripeness of the sperm, as is the rate of activation of the sperm of E. esculentus, the decrease being less in the riper sperm and absent in fully ripe sperm.

   • (c) In the presence of the egg-secretions the decrease of activity is much reduced (Gray, 1927).

   • (d) The presence of thyroxine produces effects exactly similar to those of the secretions.

   • (e) It appears that substances related to thyroxine are never more than partially effective in producing these effects.

The results of these experiments therefore confirm the conclusion that the secretions contain a body similar to thyroxine in physiological action on the sperm.

The experiments also show that there is a parallel in their action on the sperm between the effects produced by the egg-secretions and thyroxine and those of changes in the hydrogen-ion concentration of the medium. But the effects produced by these substances on one species of sperm are similar to those produced by alkalinity, and on the other to those produced by acidity.

Further, the experiments show that the oxygen consumption of fully ripe sperm of both these species is unaffected by the presence of either the egg-secretions or thyroxine¹. This result is, perhaps, surprising. It might have been expected that the secretions would have been absorbed by the sperm, whenever they were present in the medium, and would have produced their characteristic effects, whenever they were absorbed. On the contrary, as it becomes ripe, the sperm is able to produce, spontaneously and more and more completely, the effects which are produced in unripe sperm by the presence of the secretions. It seems that the only probable explanation of this result is that the ripe sperm normally contains a sufficient supply, for its activity in normal sea-water, of the substance which it otherwise receives from the secretions, and that ripening of the sperm in this character consists in the elaboration of this substance in the sperm. If the substance is related to thyroxine, which may very probably be a widespread constituent of the animal cell, this supposition is much more probable than if it were some substance peculiar to the egg.

If this is true, the meaning of the secretions in the phenomena of fertilisation, so far as the activity of the sperm is concerned, would seem to be the conversion of unripe into ripe sperm, when the sperm comes into the neighbourhood of the egg. But it seems that this phenomenon cannot be of much value in the natural fertilisation of the egg. It appears to be improbable that unripe sperm is normally shed in any quantity. However, it has also been shown that the activity of sperm which has been kept for some hours undiluted after it has been taken from the urchin, is increased by the presence of the secretions. If the secretions have a similar effect on sperm which is ageing after a long period of activity in sea-water, and the results given in the previous paper (Carter, 1930, p. 46) indicate that this may be so, the presence of the secretions may be of more definite value in fertilisation.

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