The Physiology of Sea-Urchin Spermatozoa: Catalase

The existence of catalase in the semen of Echinus esculentus was briefly mentioned in a previous paper (Rothschild, 1948 a) and a similar observation was made by Evans (1947) using the sperm or semen of Arbacia punctulata. Quantitative determinations of catalase content were not made in either case; nor was the presence of catalase in spermatozoa, as opposed to seminal plasma, established. Mammalian spermatozoa contain negligible quantities of catalase, its presence even being used on occasions as an index of bacterial contamination (Blom & Christensen, 1947). The existence of this enzyme in significant quantities in sea-urchin semen is therefore of special interest, though it must be remembered that sea-urchin and mammalian spermatozoa have somewhat different types of metabolism. The former are not motile in the absence of O₂ (Harvey, 1930) and exhibit negligible aerobic or anaerobic glycolysis (Rothschild, 1948b). The latter produce considerable quantities of lactic acid, 2 mg./10⁹ sperm/hr. at 37° C. anaerobically, and 50–90% of this figure aerobically (Mann, 1948), while movement is not dependent on the presence of O₂.

The main function of catalase is believed to be the decomposition of H₂O₂, formed during metabolism. Keilin & Hartree (1936) have suggested that catalase may have a peroxidatic function as well, in the sense of catalysing coupled oxidations of the type C₂H₅OH + H₂O₂→CH₃CHO + 2H₂O. Zeller & Maritz (1944) have described another role of catalase, in the interaction between L-ophioamino acid oxidase and its substrates, which follows different pathways in the presence and absence of catalase. In this paper, the expression ‘catalatic activity’ refers to the catalytic decomposition of H₂O₂ by catalase into O₂ and H₂O.

Evans (1947) refers to the toxic action of H₂O₂ on sea-urchin spermatozoa. His results can only be interpreted as showing that these spermatozoa are unable to decompose H₂O₂ added directly or indirectly to the external medium. This may be thought surprising as sea-urchin semen contains catalase, though H₂O₂ is also very toxic to a number of catalase-containing bacteria (Lwoff & Morel, 1942).

In order to clarify the position it is first necessary to make quantitative measurements of the catalatic activity of sea-urchin semen. Secondly, an answer must if possible be found to the question as to whether the catalase is in the spermatozoa or in the seminal plasma. This may involve a knowledge of the relative amounts of sperm and seminal plasma in a sample of semen ; the actual number of spermatozoa per ml. of semen does not by itself provide this information unless the volume of an individual spermatozoon is also known. The volume ratio obtained by centrifugation is rather inaccurate even at high centrifugal speeds (Shapiro, 1935), something which is likely to be aggravated by the long tail of the sea-urchin sperm and by the peculiar shape of the head. Catalatic activity is defined in terms of the dry weight of the material being examined. The dry weight of a unit quantity of seminal plasma, separated from semen by centrifugation, can readily be obtained. But even if the spermatozoa can be completely freed of seminal plasma by repeated washing, the dry weight of the centrifuged spermatozoa will include solids derived from the liquid used in the washing process, it being of course impracticable to use distilled water. Moreover, the process of washing sea-urchin spermatozoa may leach substances, possibly even catalase, out of or off the surfaces of the spermatozoa. This was mentioned in previous papers (Rothschild, 1948 a, b) in which it was shown that the supernatant fluid obtained by centrifuging suspensions of spermatozoa diluted with sea water and allowed to metabolize for some hours maintained the respiration of other spermatozoa suspended in it at a higher level than controls in ordinary sea water. Furthermore, during centrifugation, Androgamone I diffused out of the spermatozoa into the seminal plasma.

The problems outlined in the previous paragraph are the subject of this paper.

Semen, spermatozoa and seminal plasma of Echinus esculentus.

Measurements were made in differential manometers, The vessels were conical, with a capacity of about 17 ml., and had no centre cups or side-arms. The biological material was suspended in neutral phosphate buffer, sea water, or in some cases specifically mentioned, distilled water, 4 ml. of the suspension being placed in the manometer vessel, 0·1 ml. of H₂O₂ solution of known concentration was added to the suspension at t = o. Readings were taken at 1 min. intervals for 5 min., a later reading being made to determine the total O₂ evolved. The H₂O₂ solution was added from a glass dangling cup, capacity 0·2 ml., suspended in the main vessel and released magnetically at i = o. In a previous paper (Laser & Rothschild, 1947) an electromagnetic mixer, suitable for such purposes, but intended for measurements of O₂ uptake immediately after fertilization, was described. This device has certain drawbacks, the more important of which are: (1) the capillary tubing of the manometer has to be broken for incorporation of the mixer, and subsequently rejoined; (2) unless special precautions are taken there is a danger of asymmetrical heating effects due to the flow of current through the solenoids which produce the magnetic field; (3) unless elaborate safety devices are incorporated, there is a possibility of time-consuming accidents due to supply failures if the mixer is mains-operated. The new method of mixing H₂O₂ with the catalase-containing suspension (Fig. 1) is less complicated, cheaper, and generally superior. The glass dangling cup D has fused in it a piece of fine platinum wire terminating in a hook, H’. When the cup has been filled with the H₂O₂ solution it is hooked on to the platinum wire W, which also terminates in a hook H. The platinum wire passes up the bore of the manometer capillary, and at its distal end is joined to a piece of iron wire, I. The magnet M holds the iron wire, and therefore the platinum wire and the dangling cup, above the suspension while the manometer is being shaken, in these experiments at 170 c.p.m. with a 3·5 cm. stroke. Surface tension prevents the H₂O₂ solution splashing out of the cup even at such high shaking speeds. At t = o the release knob K is pushed in and the cup falls into the suspension in the main vessel. In practice, the shaker speed is kept at 95 c.p.m. during the 10 min. equilibration time, and as t approaches o it is gradually increased to 170 c.p.m., the index finger of the right hand being kept on the release knob. With a dangling cup of o-2 ml. capacity containing 01 ml. of H₂O₂ solution, 4 ml. of fluid in the main vessel was found to be the optimal amount for rapid mixing and diffusion, one of the limiting factors in this method of measuring catalatic activity. Perspex dangling cups, though easy to make, were found to be less satisfactory than glass ones as they tend not to sink at once after being dropped ; also, after release, they cannot be heard rattling in the main vessel.

All experiments were done at 15° C. with air in the gas phase of the manometers. The H₂O₂ solution in the dangling cups was equivalent to about 1500 μl. O₂/ml. and was prepared by diluting 95 vol. H₂O₂ with glass-distilled H₂O. The concentration of the H₂O₂ solution was checked periodically by titration with KMnO₄ in the presence of H₂SO₄ and also by measuring the amount of O₂ liberated from 0·1 ml. H₂O₂ solution when this was added to horse-liver catalase in neutral phosphate buffer in the main vessel.

For purposes of comparison, a protocol is given in an Appendix showing the catalatic activity of the writer’s blood. This, and the details given in the Results section, enable to be compared with ‘Kat.F’ (Sumner & Somers, 1943), ‘Q-Kat.’, (v. Euler, 1934), or any other method of expressing catalatic activity such as the velocity constant k₁ of Bonnichsen, Chance & Theorell (1947), to which A_c is closely related. The catalatic activity of different solutions cannot be compared purely on the basis of the dilution necessary to produce, for example, 70 μl. O₂ in the first minute, from an H₂O₂ solution containing 150 μl. O₂, as the solutions may not be comparable in regard to their content of solid material. In comparing the catalatic activity of blood and semen, therefore, attention must not only be paid to the dilution but also to the dry weight of a unit volume of the suspension used in an actual experiment.

The range over which there is a linear relationship between enzyme concentration and rate of O₂ evolution is limited. The reason for this at low enzyme concentrations is the destruction of catalase by H₂O₂ ; when the enzyme concentration is too high, the rate of O₂ evolution is affected by diffusion limitations and by the inertia of the measuring system. The amounts of semen and seminal plasma were adjusted as far as was possible so that 0₂ evolution was in the linear range.

The O₂ uptake of spermatozoa was examined in the presence of H₂O₂, neutralized hydroxylamine hydrochloride (‘hydroxylamine’) and H₂O₂ + hydroxylamine. The experiments were carried out in differential manometers = i-2, reagents being added from side-arms. The main vessels contained 2-o ml. sperm suspension and the centre cups o·2 ml. 10% KOH and filter-papers. The temperature was 15° C., the gas phase was air, and the shaking rate was 100 c.p.m. with a 3·5 cm. stroke.

Photoelectric absorptiometer (Rothschild, 1950).

The catalatic activity of semen in sea water was found to be 50 (average of 16 determinations) and of seminal plasma 210 (average of 8 determinations). From these figures the catalase content of seminal plasma appears to be four times greater than that of semen; this makes it difficult, if not impossible, to correlate catalatic activity with seminal sperm density, as the catalatic activity of the spermatozoa appears to be constantly overshadowed by that of the medium.

Estimations of catalase content involve measurements of the rate of evolution of 0₂ from H₂O₂. There is no doubt that sea water is an unsatisfactory medium in which to make such measurements on spermatozoa. Sea water, being the normal environment of ejaculated spermatozoa, is not harmful to them; consequently there is a delay before the added H₂O₂ reacts with the catalase in the spermatozoa, making the activity of semen appear lower than it really is. This difficulty does not apply in the case of seminal plasma in which there are unlikely to be any surfaces which might impede the interaction between catalase and H₂O₂. The catalatic activity of semen in neutral phosphate buffer, which is toxic to spermatozoa, was found to be twice that in sea water (Table 1).

The difference between the catalatic activity of semen in sea water and phosphate buffer is strong evidence for the existence of catalase in spermatozoa apart from its presence in seminal plasma, though phosphate buffer may increase the activity of the catalase which is present. An experiment to demonstrate this point, but comparing the catalatic activity of semen in sea water and distilled water, which is of course as toxic as phosphate buffer, is given in Table 2. The higher activity of semen in distilled water strongly suggests that catalase diffuses out of the spermatozoa (or H₂O₂ in) more readily when they are in this medium than when in sea water.

Confirmation of the existence of catalase in spermatozoa is obtained from experiments in which semen is washed with sea water (Table 3). If the apparent catalatic activity of semen were only due to seminal plasma, spermatozoa which have been once washed with sea water should have a lower catalatic activity than sperm in seminal plasma. Table 3 shows that this is not the case. The same result is obtained when the semen is repeatedly washed, by centrifugation, removal of supernatant fluid, and re-suspension to the original sperm density by addition of sea water. This procedure reduces the catalatic activity of the seminal plasma because of its dilution with sea water, but the catalase content of the final suspension, which contains sperm in sea water, is not correspondingly reduced. In some experiments, particularly with the semen of Psammechinus miliaris which were done the previous year, the reduction in catalatic activity of seminal plasma was not proportional to its dilution ; moreover, in some cases there was an increase in the activity of ‘2nd’ seminal plasma (see Table 3, V₃). Both experiments suggest that in certain conditions, catalase diffuses out of spermatozoa, or off their surfaces, into the medium. This would be of great interest if the process of centrifugation and re-suspension did not damage or kill a significant number of spermatozoa. The only quantitative test for injury or lack of injury at present available is that of 0₂ uptake; there is no doubt that 0₂ uptake is markedly reduced by centrifugation.

10⁻⁴M-hydroxylamine strongly inhibited the decomposition of H₂O₂ by semen and seminal plasma.

When H₂O₂ was added to sea water there was no evolution of O₂.

The effect of H₂O₂ is shown in Fig. 2. In this experiment, measurements of O₂ uptake were made on four identical sperm suspensions at the same time. After 10 min., during which the four suspensions respired at the same rate, 0·3 ml. sea water was added to the sperm suspension in the first vessel, curve I; 0·3 ml. sea water containing H₂O₂ to the suspension in the second vessel, final concentration 7·8 × 10⁻⁵M, curve II; 0-3 ml. sea water containing H₂O₂ to the suspension in the third vessel, final concentration 3·9 × 10⁻⁵M, curve III; and 0-3 ml. sea water containing H₂O₂ to the suspension in the fourth vessel, final concentration 4·3 × 10⁻⁴M, curve IV. The spermatozoa were able to deal with the added H₂O₂ in each case, though in the case of the highest concentration of H₂O₂, there was a pronounced shock effect, from which the spermatozoa only recovered after 20 min., as evidenced by the rate of O₂ uptake becoming similar to that in the control. Part of the depressed O₂ uptake in curve IV is due to the positive pressure in the manometer, when the added H₂O₂ is decomposed into O₂ and H₂O. This positive pressure does not account for the whole of the drop in O₂ uptake, as curve V, which is curve IV corrected for the simultaneous evolution and uptake of O₂, shows. The amount of O₂ evolved in the Exps. II and III is too small to be detected.

Hydroxylamine depresses O₂ uptake (Fig. 3) and motility. O₂ uptake was measured in three identical sperm suspensions which respired at the same rate before treatment. After 15 min., 0·3 ml. sea water was tipped into the first suspension, curve I; hydroxylamine, in seawater, final concentration 3·313 × 10⁻⁴M, was tipped into the second suspension, curve II; and hydroxylamine in sea water, final concentration 1·66 × 10⁻⁴M was tipped into the third suspension, curve III, Apart from affecting O₂ uptake, the stronger hydroxylamine solution markedly reduced the motility of the spermatozoa.

At such low concentrations, hydroxylamine is more likely to inhibit catalase than cytochrome. The cytochrome system of sea-urchin spermatozoa, might, however, be unusually sensitive to hydroxylamine ; in order to exclude this interpretation of the observed inhibition, experiments were done to test the effect of hydroxylamine, H₂O₂ and a combination of both, on the O₂ uptake of sperm suspensions. The results are shown in Fig. 4. Curve I is the control in which at t= 10 min., 0·30 ml. sea water was tipped into the sperm suspension in the main vessel. Curve II shows the effect of adding H₂O₂, final concentration 1·2×10⁻⁴M. The small positive pressure due to the evolution of O₂, or the ‘shock’ effect of adding H₂O₂, is clearly visible between t = 20 and t = 30. After this, recovery is complete, showing that the spermatozoa were unaffected by the added H₂O₂. Curve III shows the characteristic reduction in O₂ uptake on addition of hydroxylamine, final concentration 3·31 × 10⁻⁴M. Curve IV shows the effect of adding the two reagents simultaneously, the final concentrations being the same, that is, H₂O₂, 1·2 × 10⁻⁴M; and hydroxylamine, 3·31 × 10⁻⁴M. The dramatic and abrupt cessation of O₂ uptake and motility which follows this treatment could be interpreted as showing that at these concentrations hydroxylamine specifically inhibits catalase activity and not the cytochrome system in the spermatozoa. For in the presence of hydroxylamine, a concentration of H₂O₂, which is harmless alone, is extremely toxic. The possibility that in these conditions, hydroxylamine exerts a different and perhaps indirect effect cannot be completely excluded. Both in curves II and IV there is a significant delay before the H₂O₂ exerts its maximum effect, as in the experiments on the catalatic activity of spermatozoa in sea water.

On the basis of these experiments we should expect to be able to differentiate manometrically between a lethal dose of H₂O₂ and the same dose of H₂O₂ + hydroxylamine; the apparent total O₂ uptake should be lower in the former than in the latter, because of the evolution of O₂, when the catalase is not inactivated. An experiment to test this is shown in Fig. 5. Curve I is the control in which 0·3 ml. sea water was added to the sperm suspension in the main vessel at t = 10. In curve II hydroxylamine in 0·3 ml. sea water was added, the final concentration being 3·31 × 10⁻⁴M. In curve III, hydroxylamine, final concentration 3·31 × 10⁻⁴M, and H₂O₂, final concentration 2·9×10⁻⁴M, were added simultaneously. In curve IV 0-3 ml. sea water, containing H₂O₂, final concentration 2·9 × 10⁻⁴M, was added at i=io, the total O₂ consumed by the sperm in the presence of H₂O₂ alone was apparently about 4 μl. less than when H₂O₂ and hydroxylamine were added together.

The volume of a sea-urchin spermatozoon is about 15 μ³. This figure is only approximate and is obtained by graphical integration of drawings based on microphotographs. It assumes that the sea-urchin spermatozoon, unlike that of the bull, has axial symmetry, i.e. that the correct volume of the spermatozoon is obtained by estimating the volume of the solid formed by rotating the spermatozoon about its long axis.

The catalatic activity of seminal plasma in phosphate buffer is about twice that of semen in the same medium. The experiments on the increase in seminal catalatic activity in distilled water, and those showing that washed semen does not lose its ability to decompose H₂O₂ prove without reasonable doubt that both sea-urchin spermatozoa and seminal plasma contain catalase. The question can, however, be examined from an entirely different point of view, by considering the relative volumes of sperm and seminal plasma in a sample of known catalatic activity and sperm density. In the experiment in Table 3, there were 2·85 × 10⁻¹⁰ sperm/ml. semen. The volume of a spermatozoon is about 15 μ ³0·30 ml. semen therefore consisted of 0·128 ml. spermatozoa and 0·172 ml. seminal plasma. 0·30 ml. seminal plasma decomposed 52 μ.1. O₂ from an H₂O₂ solution containing 155 μl. O₂ in 1 min. 0·172 ml. seminal plasma would therefore have decomposed 30 μl. O₂ in the same time. But in fact this amount of seminal plasma, in the presence of 0·128 ml. spermatozoa, produced 92 μl. O₂ in i min., showing that the sperm contributed to the catalatic activity of the semen.

Mention has already been made of the difficulty, if not the impossibility of measuring the catalatic activity of spermatozoa as opposed to semen or seminal plasma. It is therefore equally difficult quantitatively to relate the catalatic activity of semen and washed spermatozoa. Table 3 suggests that if it were possible to measure it, the catalatic activity of spermatozoa might well be much higher than that of seminal plasma. It is evident from this table that the catalatic activity of washed sea-urchin spermatozoa requires further study, though such a study presents several difficulties.

The low concentration of hydroxylamine which affects the O₂ uptake and motility of sea-urchin spermatozoa suggests that, in these conditions, catalase rather than cytochrome is attacked by this inhibitor. This view is strengthened by the observation (Rothschild, 1948b) that a large reduction in O₂ uptake can be effected by CO in the dark, which prevents the reoxidation of cytochrome oxidase, without motility being correspondingly reduced. This indicates that, although sea-urchin sperm motility is dependent on the presence of O₂, only part of the O₂ consumption of the spermatozoa is directly concerned with the energy requirements of movement. Hydroxylamine, on the other hand, reduces motility even when it inhibits O₂ uptake to a relatively small extent. If the view is accepted that at low concentrations hydroxylamine combines with catalase, this observation shows that catalase has a functional role in sea-urchin sperm metabolism. The experiments showing that H₂O₂ is harmless by itself but highly toxic in the presence of hydroxylamine prove the first point, that the effect of hydroxylamine in these conditions is to inactivate catalase ; they do not unequivocally prove that sperm metabolic processes normally result in the production of H₂O₂ which is decomposed by catalase. Catalase may have some role other than decomposing H₂O₂; if this is so, when this role is interfered with by hydroxylamine; O₂ uptake and motility may be reduced for some reason other than the accumulation of H₂O₂. Even if this is true, catalase has been shown to be available for the decomposition of H₂O₂ as well as to perform this other hypothetical function. Though there is a case for the view that the function of catalase in sea-urchin spermatozoa is to decompose H₂O₂ formed during metabolism, the only way of proving this explicitly would be by a chemical demonstration that H₂O₂ accumulates in the presence of hydroxylamine.

Evans (1947) observed that heavily irradiated sea water harmed the spermatozoa of Arbacia punctulata. His interpretation was that the irradiation produced H₂O₂ which, at concentrations of less than 0· 5 part per million, was toxic to the spermatozoa. This result might be considered surprising in view of the catalase content of seaurchin spermatozoa, which Evans also observed (see Lwoff & Morel, 1942).

A further surprising feature of these experiments was that the toxic effects of H₂O₂ were neutralized both when catalase and heat-inactivated catalase were added to the suspensions. The differences between Evans’s results and those described here may in part be due to differences in technique. In one case fertilizing capacity and survival times were observed; in the other O₂ uptake was measured. There are certain disadvantages in adding H₂O₂ to manometers which are subsequently shaken, but the experiments described in this paper are not affected by them, because the H₂O₂ was not decomposed in control experiments without spermatozoa and because the H₂O₂ experiments were combined with ones in which hydroxylamine was added at the same time to inhibit catalase, when the toxic action of H₂O₂ was plainly visible.

From the data in the Results section, a rough calculation can be made of the number of catalase molecules per spermatozoon. According to Green (1940), one mole of catalase decomposes 4·2 × 10⁻⁴ moles of H₂O₂ per second at o°C. As from Table 3, 8·55 × 10⁻⁹ washed spermatozoa produced 90 μl. O₂ in 1 min., each spermatozoon contains less than 500 molecules of catalase.

Dry weight of 1 ml. blood (R), 0·2293 g.

Dilution of blood for manometric experiment, 1 ml./1667 ml. neutral phosphate buffer.

Main vessel, 4·0 ml. blood phosphate mixture.

Dangling cup, 0·1 ml. H₂O₂ solution, containing 146 μl. O₂.

O₂ produced in 1 min. after mixing, 87 μl.

A_c, 6800.