The Respiration of Sea-Urchin Spermatozoa

Gray (1928) found that dilute suspensions of sea-urchin spermatozoa in sea water consumed more oxygen during their active life and respired at a higher rate, per unit quantity of spermatozoa, than dense suspensions. These phenomena are together known as the Dilution Effect. In this paper the first of these two characteristics is called Dilution Effect I, while the increased rate of 0₂ uptake by dilute suspensions is called Dilution Effect II. The term Dilution Effect is sometimes used in the field of mammalian sperm physiology, with a different meaning ; for in this case, it refers to the decline in the viability of sperm suspensions when diluted too much. In such circumstances mammalian spermatozoa may lose some substance to the suspending medium, with concomitant impairment of motility (Emmens & Swyer, 1948).

Such expressions as ‘dense suspension’ and ‘dilute suspension’ require precise definition. The most satisfactory way of expressing sperm concentrations is in terms of the number of spermatozoa per ml. of suspension. Other methods, involving measurements of the amount of nitrogen in the suspension, or the dry weight of the spermatozoa, are not so satisfactory, for reasons that have been discussed in a paper on counting spermatozoa (Rothschild, 1950a). Suggested definitions of ‘very dense ‘, ‘dense’, and ‘dilute’, are as follows (Rothschild & Tuft, 1950):

These definitions apply to the semen of Echinus esculentus, in which the concentration of spermatozoa is about 2 × 10¹⁰/ml. The three concentrations given above are therefore equivalent to the following dilutions of the original semen with sea water: 1 : 4, 1 : 50 to 1 : 20 and 1 : 50. The range of concentrations between 5 × 10⁹ and 10⁹ sperm/ml. was deliberately left undefined.

Dilution Effect II was observed between suspensions containing 8·00 × 10⁸ and 5·58 × 10⁸ sperm/ml. by Rothschild & Tuft (1950). Gray (1928), measuring sperm concentrations in terms of mg. N/ml. suspension, which have been converted to sperm/ml. on the basis of his figures and the average value of 2 × 10¹⁰ sperm/ml. in undiluted semen, observed Dilution Effect II in the following pairs of experiments (the figures are necessarily approximate) :

In all these, the sperm suspensions were either ‘very dense’ or between ‘dense’ and ‘very dense’. When a comparison was made between suspensions containing 8 × 10⁸ and 4 × 10⁸ sperm/ml. no Dilution Effect II was observed. Barron, Gasvoda & Flood (1949) also noted Dilution Effect II in the spermatozoa of Arbaciapunctulata. They state that the maximum ⁠,19·6, was attained in this species at a dilution of 1 : 200. These experiments were carried out on semen which was centrifuged for 10 min. at 2000 r.p.m., the supernatant seminal plasma being removed. After this, ‘the remaining sperm was brought to the desired dilution starting with a stock dilution of 1 : 10 or 1 : 20’ (p.44). This probably means that a dilution of 1 : 10 in these conditions corresponds to a dilution of the original semen of 1 : 15 or 1 : 20. Apart from the difficulty in knowing the true sperm concentrations in these experiments, no details of temperature nor the method of effecting the dilution are given. This latter point is important, for if on dilution there is a sudden burst of respiration, dilution must be performed within the measuring apparatus, after temperature and gas equilibration. Otherwise the burst of respiration will be lost during the 15 min. or so needed for dilution and equilibration. The fact that it would be difficult if not impossible to achieve a dilution of 1 : 1600 within an ordinary Warburg manometer may explain why these workers found a of zero at this dilution. During the 15 min. before measurements could be begun, the suspension finished its O₂ consumption.

The Dilution Effect has been said to show first, that when dilute, sea-urchin spermatozoa have more room to move and therefore move more, consuming more O₂ in the process (see, for example, Barron et al. 1949). Secondly, that these spermatozoa do not die or senesce purely because of lack of fuel (endogenous substrate), but also because of some form of self-intoxication, using this word in a very general sense. There are certain difficulties in fitting these facts into a consistent picture of sea-urchin sperm metabolism, and it therefore seemed advisable to re-examine the basic features of their respiration under rigorous conditions. The experiments described in this paper were done with that object and, apart from clarifying some of the issues mentioned above, they may facilitate interpretation of some of the anomalous results obtained in the study of sea-urchin sperm metabolism, which have recently been tabulated in a review (Rothschild, 1951).

Semen of Echinus esculentus. This consists of spermatozoa and seminal plasma. The latter is a viscous transparent fluid, sometimes yellowish and sometimes pinkish in colour. It contains fats, about four times as much potassium as sea water or coelomic fluid (Rothschild, 1948 a), and in certain conditions, the substance Androgamone I, called A. I (Hartmann, Schartau, Kuhn & Wallenfels, 1939), which is not a protein. The semen was not washed before use for the following reasons. Washing consists in centrifuging the semen, removing the supernatant seminal plasma, and resuspending the spermatozoa in sea water, the procedure usually being repeated three times. It damages or reduces the viability of the spermatozoa if done thoroughly (see later), while if not done thoroughly, as in the experiments of Barron et al. (1949), when the semen was centrifuged for 10 min. at 2000 r.p.m. and not re-suspended until required for an experiment, there is little point in the procedure as the objective—to remove all the seminal plasma—is not achieved.

Photoelectric absorptiometer. This method of counting spermatozoa, which has been described in detail (Rothschild, 1950 a), involves obtaining a relationship between the density of a suspension, determined with a haemocytometer, and the absorptiometer reading for the same suspension.

The results described later are closely bound up with the question of adequate O₂ saturation of the sperm suspensions in the manometer vessels. The apparatus used for measuring O₂ uptake must therefore be described in some detail. Measurements were made with Warburg manometers, volume to manometer fluid approx. 15 ml., and differential manometers of about the same volume. The vessels were of conventional conical shape. They contained about 3, 2, 1·5 or 1 ml. of sperm suspension in different experiments. The shaking rate was 94 or 120 c.p.m., with a 4 cm. stroke in each case. The centre cup contained 0·1 ml. 10% KOH and the usual flowered filter-papers (Whatman no. 40) protruding above the top of the cup. The gas space contained air and the bath temperature was 15·0’³ C. The manometers were calibrated by the standard Hg method and also by liberating a known amount of N₂ in the flasks by the reaction 2KIO₃ + 3N₂H₄→2KI + 3N₂ + 6H₂O. The solubilities of N₂ and O₂ in water are sufficiently low and similar to make the error due to N₂ being liberated instead of O₂ negligible (Dixon, 1943).

Total O₂uptake of dense and dilute suspensions, Dilution Effect I. Previous experiments in which Dilution Effect I was observed involved a comparison of the O₂ uptake of two suspensions, one containing about 3 × 10⁹ sperm/ml. and the other 8 × 10⁸ sperm/ml. One might suspect that O₂ saturation was a limiting factor in the O₂ uptake of the denser suspension, particularly as 6 ml. of suspension was used in the manometer flask. This was shown to be the case by the following experiment. A sample of semen containing 1·78 × 10¹⁰ sperm/ml. was diluted six times with sea water and different amounts, 3, 2, 1·5 and 1 ml. of this suspension placed in four Warburg manometers. The surface area exposed to the air in the manometer vessel increases as the amount of suspension is decreased, while the thickness of the suspension naturally decreases. If O₂ saturation is a limiting factor, we should expect that the O₂ uptake per unit quantity of spermatozoa would be highest in the vessel containing the smallest quantity of suspension. This method of testing the adequacy of O₂ saturation is more satisfactory than the conventional one of varying the shaking speed (although this was done as well), for two reasons. First, it appears to be more sensitive (Table 1); secondly, high shaking speeds may damage seaurchin spermatozoa (Rothschild, 19480). The results of this experiment are shown in Table 1. Table 1 shows that at this sperm concentration, 3 × 10⁹/ml., O₂ saturation is a seriously limiting factor in the O₂ uptake of the suspension. This applies in all four cases as the O₂ uptake in vessel 4 increased when the shaking speed was increased. Although unimportant in this context, the explanation of the lack of increase in O₂ uptake in vessels 1-3 at the increased shaking speeds is probably that even in these conditions the ‘internal resistance’ of the larger volume of suspension prevented adequate mixing. In vessel 3, the decline in O₂ uptake may have been arrested at the higher shaking speed. Further experiments, similar to that described in Table 1, showed that the minimum permissible dilution, using 2 ml. of suspension, is 1 : 25, that is a sperm density of about 8 × 10⁸/ml. In fact, it is safer to use concentrations between 1 : 30 and 1 : 50. However, to investigate Dilution Effect I, concentrations of 1 : 25 and 1 : 50 were used, to approximate as nearly as possible to the conditions in previous experiments. The details of an experiment on Dilution Effect I are shown in Table 2, and the results are shown in Fig. 1. The third vessel, containing sea water and seminal plasma obtained by centrifuging semen for 10 min. at 2000 g. in an angle centrifuge, was a control to obviate the effects of O₂ uptake (if any) of seminal plasma, and to try and counteract the influence of bacterial metabolism towards the end of the experiment, which lasted some 18 hr. Seminal plasma had no O₂ uptake, though there was a small bacterial effect towards the end of the experiment; this has been appropriately subtracted from the values for sperm O₂ uptake obtained in the experimental vessels. At t = 0, the contents of the side arms were transferred quantitatively into the sea water in the main vessels. In a normal manometric experiment, when a reagent is added from the side arms, the latter need only be washed into the fluid in the main vessel three times at the most. In this experiment, however, it was essential for all the semen, which is very viscous, to be mixed thoroughly with the sea water in the main vessel. To ensure this, the side arms were washed into the main vessels twenty times, which took about 2 min. The experiment shows that the total O₂ consumed, per unit quantity of sperm, by the denser suspension, so far from being less than that consumed by the dilute suspension, is in fact somewhat greater. There are some difficulties in determining the end-point because of autolytic and bacterial O₂ uptake after the spermatozoa have become motionless. These can be overcome in two ways. First, by simultaneously running two vessels whose contents and treatment are identical with those of the experimental vessels, from which samples are periodically taken for microscopic examination. By this method the time at which 95 % of the spermatozoa in the dilute suspension were dead could be easily determined. It is possible to predict when this will occur, quite accurately, from examination of the O₂ uptake curve. Secondly, soon after the spermatozoa have become motionless, there is a marked and characteristic inflexion in the curve (Fig. 1,1), due to the onset of bacterial and autolytic O₂ uptake. Although all the spermatozoa in a suspension do not die at the same time, and the autolytic O₂ uptake of those which die early contributes to the ‘true’ O₂ uptake, the majority become motionless towards the end of the life of the suspension as a whole. This type of experiment was repeated at lower sperm densities, with the same result (Fig. 2). At such sperm concentrations Dilution Effect II is barely perceptible. The higher total O₂ uptake of the denser suspension is, however, visible.

The low rate of O₂ uptake of a 1 : 6 suspension and its lower total O₂ uptake are due to inadequate O₂ saturation, a high proportion of the spermatozoa in the suspension not respiring at all because of zero or nearly zero O₂ tension in perhaps two-thirds of the suspension. The experimental conditions are not entirely clear in the paper by Barron et al. (1949), but it seems probable that the low ’s observed at high sperm densities, 1·7 and 3·6 at dilutions stated to be 1 : 10 and 1 : 30, had the same cause. There is of course no way of telling whether a biological tissue in a manometer is, or is not, suffering from inadequate O₂ saturation unless the shaking rate or the thickness of the material is varied, as the measured rate of O₂ uptake may satisfy the accepted requirements for the relationship between shaking rate and O₂ uptake, because O₂ saturation is inadequate.

These experiments show that Dilution Effect I, the increased total O₂ uptake of dilute suspensions, does not exist when the experimental conditions are physiologically satisfactory. When the conditions are not satisfactory the effect is due to spermatozoa suffering from anoxia.

Dilution Effect II. There are certain difficulties in the concept that the increased rate of O₂ uptake by dilute sperm suspensions is a result of the spermatozoa having more room to move in such suspensions. First, the energy expended by a spermatozoon is pre-supposed to be inversely proportional to the constraining forces of the system. Secondly, spermatozoa may move faster when diluted, but it does not follow that faster movement is a direct result of dilution. As chemical changes must precede the alterations in the structure of the sperm tail which produce the faster movement, dilution may affect some aspect of metabolism first and this in turn may determine the subsequent movements of the spermatozoon. A different but plausible explanation is suggested by the concept, due to Hartmann et al. (1939),^* that A. I, which inhibits motility, diffuses out of sea-urchin spermatozoa. They held that lack of motility in semen was caused by this substance. This is not true in the case of E. esculentus or Psammechinus miliaris, but is true in salmon semen (Runn-strom, Lindvall & Tiselius, 1944; Rothschild, 1951). If A. I were present in seminal plasma, the decrease in its concentration on dilution of a sperm suspension and consequent reduction of the inhibitory action of seminal plasma might explain the increased O₂ uptake of dilute suspensions. This possibility is easily disposed of by the experiment shown in Table 3, in which a comparison was made between the respiration of spermatozoa in sea water containing different amounts of seminal plasma. High-speed centrifuges must be used to measure directly the ratio of seminal plasma to semen in a sample. As these were not available previously, the volume of a spermatozoon was estimated by graphical integration of oil-immersion phasecontrast photo-micrographs, and found to be 15-20 μ³ (Rothschild, 1950b), the uncertainty being mainly due to difficulties in measuring the radius of the sperm tail. Through the kindness of Dr R. Markham, the Molteno Institute, Cambridge, the figure of 15-20 μ³ has been compared with estimates based on centrifugation. A sample of semen was centrifuged for about 45 min. at 23,000 g. in an ultracentrifuge, the boundary between the spermatozoa and the seminal plasma being photographed when it had come to rest. The seminal plasma occupied about 47 % of the volume of the sample which contained 2·84 × 10¹⁰ sperm/ml. A simple calculation shows that the volume of each spermatozoa is therefore about 18 μ³. This will be discussed later in another context. In the sample in Table 3, the relative volumes of spermatozoa and seminal plasma per ml. semen were therefore about 0·35 ml. spermatozoa and 0·65 ml. seminal plasma. To bring the concentration of seminal plasma in a 1 : 50 sperm suspension up to the amount which a 1 : 25 suspension would contain, 0·013 ml-seminal plasma must be added per ml. sperm suspension. The experiment in Table 3 therefore shows that dilution of seminal plasma is not responsible for Dilution Effect II.

The next possibility to be investigated was that although no mutually inhibitory substances diffuse out óf spermatozoa (Rothschild, 1948 b), a substance might diffuse out of diluted spermatozoa, or off their surfaces, causing the affected spermatozoa to respire at a higher rate. Such an effect could be likened to the inactivation of a ‘respiratory Pasteur enzyme’ or the loss of some respiratory regulator. To test it, semen was diluted 1 : 50 with sea water and allowed to respire for 45 min. The suspension was then divided into four equal samples, 1, 2, 3 and 4. Sample 1 was centrifuged for 10 min. at 1100 g. and the spermatozoa were re-suspended in their own supernatant fluid, composed of sea water and seminal plasma. Sample 2 was centrifuged for 10 min. at 1100 g., after which the supernatant fluid was removed, sea water was added, up to the original volume, and the spermatozoa were resuspended. Sample 3 was treated in exactly the same way as sample 2 except that after the supernatant fluid had been removed, half the quantity of sea water was added, so that after re-suspension the sperm density in sample 3 was double that in sample 2. Sample 4 was not centrifuged. Aliquots from all four samples were put in manometers and their O₂ uptake measured. In an experiment of this type, the decantation procedure cannot be assumed to achieve the desired result of doubling the sperm density, because variable numbers of spermatozoa are removed with the supernatant fluid. After the procedure outlined above, sperm counts were therefore made on the suspensions before they were put into the manometers. O₂ uptake was scaled according to these counts. The results of one of these experiments is shown in Table 4.

This experiment illustrates the following points :

• Centrifugation (c.f. V₁ and V₄, Table 4) markedly reduces the O₂ uptake of sea-urchin spermatozoa. This has been mentioned before (Rothschild & Tuft, 1950), but no quantitative information was given. Other workers, who have used washed sea-urchin spermatozoa in respirometric experiments, have not mentioned this fact.

• Seminal plasma has a protective effect on the spermatozoa (cf. V₁ and V₂ or V₂). This was known before (Hayashi, 1946; Rothschild, 1948b).

• There is no Dilution Effect II.

This experiment was repeated a number of times and the results are shown in Table 5. As the object was to confirm or reject the hypothesis that Dilution Effect II is caused by the diffusion of a regulatory substance out of spermatozoa in dilute suspensions, the controls, that is V₁ and V₄ in Table 4, were not always done and are omitted from the table. These experiments show that, under these conditions, there is a marked reduction in Dilution Effect II, which may mean that during the course of metabolism, some substance concerned with the regulation of respiration diffuses out of the spermatozoa. They do not, however, prove that this is the cause of Dilution Effect II and it is possible by a different experiment to show that another explanation must be sought. If the sperm suspensions are only allowed to respire for 10 min. before the centrifugation and concentration procedure, Dilution Effect II is much more pronounced (Table 6). This again suggests that 40 min. incubation in dilute suspension is associated with the loss of a regulatory substance from the spermatozoa but confirms that this is not the cause of Dilution Effect II. If the change on dilution were reversible, we should expect that after re-concentration in the way described above, there would be the same difference between dense and dilute suspensions, as regards O₂ uptake per unit quantity of spermatozoa, as there is before this treatment. Protective substances, such as albumin or seminal plasma, probably act by reversing this effect or preventing its occurrence. The experimental conditions preclude the possibility that the dense and dilute suspensions were in different physiological conditions as a result of treatment, because both were treated in the same way until the moment of re-suspension. Though these experiments do not establish the cause of Dilution Effect II, they indicate that in other respects, the phenomena associated with the dilution of sea-urchin spermatozoa have much in common with those observed in mammalian spermatozoa, in which it is well known that in the absence of protective substances, dilution is followed by the loss of material from the spermatozoa and by impairment of viability.

If Dilution Effect II, instead of being thought of as the increased rate of 0₂ uptake per unit quantity of spermatozoa in the more dilute suspension, is considered as the decreased rate in the more dense suspension, certain obvious questions come to mind. First, is some substance missing from the medium, or in short supply, with the result that the respiration of more dense suspensions is reduced? Secondly, can Dilution Effect II be artificially produced in suspensions which normally do not exhibit the effect, such as in the experiment shown in Fig. 2. Some preliminary investigations on the effect of copper and zinc on sea-urchin sperm respiration were recorded in a previous paper (Rothschild & Tuft, 1950). These experiments have been extended and it has been found possible to reverse Dilution Effect II by addition of CUC1₂2H₂O to the suspending medium. An example of an experiment in which this was done is shown in Fig. 3. In this experiment, which was done in duplicate, the main vessels of the manometers contained, (I) 2 ml. sea water; (II) 2 ml. sea water; and (III) 2 ml. sea water containing CUC1₂2H₂O, final concentration 1 ·1 p.p.m. The side arms contained 0 ·2 ml. of semen, density 2 ·35 × 10¹⁰ sperm/ml., diluted 1 : 2·5 with sea water in side arms (II) and (III), and 0·2 ml. of the same semen, diluted 1 : 5 with sea water, in side arm (I). The slight initial dilution of the semen was to facilitate the quantitative transfer of the side arms to the main vessels, this being done by two people. The final sperm densities in the vessels were therefore: 4·27 × 10⁸; V_II 8 ·54 ×10⁸ ; V_III, 8 ·54 ×10⁸. Fig. 3 shows that Dilution Effect II can be reversed by the addition of copper to sea water. There is a slight decline in the reversal during the second hour, which may be due to the precipitation of copper out of the sea water.

Dilution Effect II can be made to occur when the densities of the sperm suspensions are such that the effect is scarcely visible in the absence of special treatment. The reagent used to achieve this was sodium diethyldithiocarbamate (DDC), which inhibits reactions catalysed by Cu⁺⁺ ions and copper-protein enzymes such as polyphenol oxidase. There are certain problems connected with its use (Keilin & Hartree, 1940, 1949), which are discussed later. One of these concerns the possibility that DDC exerts a narcotic action on the biological material ; experiments have been done to investigate this possibility. Fig. 4 illustrates an experiment in which Dilution Effect II was imperceptible in two suspensions containing 8·87 × 10⁸ sperm/ml. sea water and 4 ·44 × 10⁸ sperm/ml. sea water (V₁ and V₂). When two identical suspensions, V₃ and V₄, were made up in sea water containing DDC, final concentration 3 ·64 × 10⁻⁵ M, the reduction in O₂ uptake of the denser suspension was 62 % while the reduction in O₂ uptake of the more dilute suspension was only 36%, in both cases after 90 min. More interesting still is the fact that in the untreated suspensions, V₁ and V₂, there was no Dilution Effect II, whereas in the treated suspensions, V₃ and V₄, there was a difference of 39% between the more dense and dilute suspensions after 90 min. respiration. If the inhibition of O₂ uptake were due to a narcotic action of DDC, or to the inhibitory effect of tetraethyldithiocarbamyl disulphide, a cytochrome c-catalysed oxidation product of DDC (Keilin & Hartree, 1940), one would not expect the percentage inhibition to vary so greatly according to the density of the suspension. The possibility that DDC does not act as a chelating agent, but as a narcotic, or in the way discovered by Keilin & Hartree, was investigated in another way, by varying the DDC concentration in the medium. The results of these experiments are shown in Table 7, which show without reasonable doubt that in these experiments DDC was acting as a chelating agent. There is, in fact, a suggestion that at the higher concentrations, DDC may have been utilized as a substrate by the spermatozoa, apart from exerting an inhibition.

Concentrations of CUC1₂2H₂O. Previously (Rothschild & Tuft, 1950), the difficulties in knowing the true concentrations of copper salts in sea water were emphasized. The values given were said to be almost certainly too high. The same applies in this paper though it has been found that if the solutions are made up immediately before the experiment, lower concentrations have the same stimulating effect on sperm O₂ uptake as higher ones made up some time before the experiment.

The experiments described in this paper enable considerable simplifications to be made in a description of sea-urchin sperm respiration and senescence. Furthermore, it now appears that the main difference between them and mammalian spermatozoa is that the former are almost entirely aerobic, while the latter, by virtue of their ability to break down fructose into lactic acid (Mann, 1946), are viable and can move in the absence of O₂. Both are adversely affected by too great dilution unless protective substances are added to the diluent. There is now no evidence to support the view that sea-urchin spermatozoa in relatively dense suspensions suffer from some form of auto-intoxication, either of an intracellular or extracellular nature. Movement stops when the endogenous substrate or some essential link in the metabolic processes is exhausted. The view that the total O₂ uptake, per unit quantity of spermatozoa, is higher in dilute than in dense suspensions, must be revised provided oxygen saturation is adequate in both cases. Because of their high ⁠, which may be more than 30 at 15^o C., inadequate oxygen saturation is a danger in all manometric experiments involving sea-urchin spermatozoa, unless the usual tests are made, or sperm counts show that the sperm density is less than 10⁹/ml. For example, in a recent and interesting paper on the effects of egg jelly and Ca⁺⁺ ions on the O₂ uptake of sea-urchin spermatozoa, Vasseur (1950, p. 393) used what he calls ‘concentrated “dry” sperm… collected in the usual way by filtering through bolting silk’, diluted 1 : 6 with sea water, in conical Warburg manometer vessels. The volumes of the vessels are not given. The semen of Echinus esculentus and Psammechinus miliaris was used. In the case of the former a semen dilution of 1 : 6 corresponds to a sperm density of about 3 ·3 × 10⁹/ml. Leaving aside the question of copper or zinc effects, this sperm density is undoubtedly too high for adequate oxygen saturation with 2-4 ml. suspension in the manometer vessels. This is confirmed by the increase in O₂ uptake when sea water was added to the suspensions as a control. The semen of P. miliaris has a higher sperm density than that of E. esculentus. Inadequate O₂ saturation cannot of course account for all of the increase in O₂ uptake Vasseur observed on addition of egg jelly and Ca++ ions. From what has been said above, the importance of making sperm counts is obvious.

The experiments involving the addition of CUC1₂2H₂O and DDC to sperm suspensions do not perhaps prove that the increased rate of O₂ uptake, per unit quantity of spermatozoa, by dilute suspensions, or more accurately, the decreased rate of O₂ uptake by dense suspensions, is due to the quantities of copper or zinc in sea water being marginal for these concentrations of spermatozoa. The experiments are consistent with this explanation, while for reasons that have already been given (p. 426), a metabolic interpretation of Dilution Effect II is needed. In undiluted semen, however, close packing undoubtedly interferes with sperm movement, when this is induced by an increase in O₂ tension. The O₂ uptake cannot be measured manometrically in these circumstances, though it might be possible to make measurements with an oxygen electrode. A quantitative examination of the possibility that the concentration of copper in sea water is marginal for dense suspensions has recently been made by Barnes & Rothschild (1950). However, the question probably does not arise in nature as the density of spermatozoa in the neighbourhood of a spawning sea-urchin is unlikely to be greater than 5 × 10⁸/ml. and usually must be much less.

Considered by itself, the stimulating action of CUC1₂2H₂O might be held to lend support to a hypothesis, put forward by Barron, Nelson & Ardao (1948), to the effect that sea-urchin spermatozoa contain two kinds of—SH groups, soluble ones which regulate respiration, and fixed ones, present in the protein moiety of enzymes. Low concentrations of—SH reagents are presumed to combine only with the first kind, causing an increase in respiration; higher concentrations combine with fixed—SH groups, with consequent inhibition of respiration. It is not obvious how the inhibition of O₂ uptake by diethyldithiocarbamate can be fitted into this picture. Moreover, in expounding this hypothesis Barron et al. make no reference to Dilution Effect II, the important feature of which is that the respiration of sea-urchin spermatozoa varies according to the density of the suspension. In another paper, however, on the effect of nitrogen mustards on sea-urchin spermatozoa (Barron, Seegmiller, Mendes & Narahara, 1948, p. 270), it is stated that ‘the results seem to depend or vary with the concentration of sperm’. Apart from necessitating further work, the resolution of these differences in interpretation depends on future experiments being carried out under clearly defined conditions, an indispensable prerequisite of which is a knowledge of the number of spermatozoa in the experimental suspension.

In conclusion, if the ‘trace metal’^* explanation of Dilution Effect II is accepted, another distinction between mammalian and sea-urchin spermatozoa becomes resolved, for one difference between dilute and dense suspensions of sea-urchin spermatozoa is due to the medium and not the spermatozoa. Barron et al. (1949) found that the O₂ uptake of sea-urchin spermatozoa was markedly lower in artificial than in natural sea water, which may have the same explanation. In his original paper on this subject, Gray (1928) observed that other cases are known in which the biological activity of cells or organisms is increased by a diminution of population density. In some of these, competition for trace metals in the medium is a possible explanation which might be worth exploring.

As this is the first time that the total O₂ consumption of a known number of seaurchin spermatozoa has been determined, it is of interest to relate this figure to the breakdown of endogenous substrate, assumed, purely for the purpose of the argument, to be ‘glucose’. In the experiment illustrated in Fig. 1, 7 ·40x10⁸ spermatozoa (2 ml. of a suspension containing 3 ·70 × 10⁸ sperm/ml.) consumed 550 pl. O₂ in 400 min. If we further assume that the average O₂ uptake of each spermatozoon was 550 ÷(7 ·40 × 10⁸) pl. O₂, each spermatozoon must break down about 10⁻⁶pg. of ‘glucose’ in 400 min. This follows from the fact that the breakdown of 1 mole (180 g.) of ‘glucose’ is associated with the disappearance of 22 ·4 × 6 1. of O₂. The volume of the middle piece of a spermatozoon of E. esculentus is not more than 5µ³. If the whole of the middle piece contained ‘glucose’ solution, this solution would have to be about 20 % to provide the necessary substrate. This concentration is rather high and suggests that the substrate may not be a carbohydrate and therefore that the respiratory quotient may be lower than one.

I am much obliged to Prof. J. Gray, F.R.S., for reading the manuscript of this paper.