The Physiology of Sea-Urchin Spermatozoa: The Nature and Location of the Endogenous Substrate

Sea-urchin spermatozoa, unlike those of mammals, do not obtain the energy necessary for movement by the enzymatic breakdown of substrates in the external medium. The evidence for this view is twofold : first, these spermatozoa are capable of prolonged activity when suspended in pure sea water; secondly, there is no obvious substrate in the normal external medium, seminal plasma diluted with sea water. Until recently the breakdown of intracellular carbohydrate has been assumed to provide the energy for movement. This assumption is mainly based on measurements of the respiratory quotient by Barron & Goldinger (1941), Hayashi (1946) and Spikes (1948, 1949). These authors do not say in their papers how they measured the respiratory quotient. Dr E. S. G. Barron was kind enough to tell one of us (R.) that he used the direct method; Dr Spikes says in his Ph.D. Thesis (1948) that he also used the direct method. In the absence of further information, Hayashi is assumed to have done the same. Conclusions based on this method, when the medium in which the biological material is suspended is weakly buffered and contains bicarbonate, are of questionable value, as the pH becomes widely different in the two suspensions during measurements. In addition, the possibility that CO₂ may influence sea-urchin sperm metabolism must be considered, if only because the respiration of sea-urchin eggs is higher when measured by Warburg’s indirect method than when the direct method is used (Laser & Rothschild, 1939). The only other evidence for carbohydrate being the endogenous substrate is Spikes’ statement (1949) that glycogen-like material disappears from the spermatozoa of Strongylo-centrotus purpuratus and Lytechinus pictus during ageing. At the beginning of the one experiment quoted, the content of glycogen-like material was 29·0 mg./g. dry weight of spermatozoa, while after 12 hr. ageing at 19° C., this had fallen to 8·5 mg./g. dry weight. No figures for oxygen uptake during the ageing period are given, though this can be estimated from information in Spikes’ Thesis.

Some calculations were recently made regarding the amount of glucose which would have to be present in the middle-piece of a sea-urchin spermatozoon to account for their observed respiration (Rothschild, 1950). The amount was found to correspond to a 20% solution, which seems very high. Subsequently, the carbo-inórate content of spermatozoa and seminal plasma of Echinus esculentus was measured (Rothschild & Mann, 1950). The glycogen-like material present in 100 ml. spermatozoa amounted to 110 mg. reducing sugar liberated by acid hydrolysis; of this only 40 mg. was glucose, while the seminal plasma contained little free reducing sugar and no glycogen-like material, though 200 mg. % of some anthrone-reactive carbohydrate, possibly a mucopolysaccharide, were present. The low concentration of sugar in the spermatozoa and the high value required to account for their respiration suggest that carbohydrate is not the principal endogenous substrate of the sea-urchin spermatozoon. The experiments described in this paper were designed to test this possibility. The methods adopted were to measure the oxygen uptake of a suspension of sea-urchin spermatozoa in sea water and the disappearance of glycogen and phospholipid in the same sample, during some 400 min. movement and respiration. The spermatozoa were also examined histochemically and with the electron microscope.

The semen of E. esculentus was obtained by the method of Rothschild & Tuft (1950), and diluted to the required density with sea water containing 50 units penicillin/ml., the penicillin, which has no effect on sea-urchin spermatozoa, being added to delay bacterial contamination (Tyler & Rothschild, 1951). Sperm counts were made with a Fuchs-Rosenthal haemocytometer, the standard error being about 5 %.

O₂ uptake was measured in Barcroft-Warburg manometers with conical flasks, capacity about 15 ml. 3 ml. of the sperm suspension was placed in the flasks and 0·2 ml. 10% KOH and filter-papers in the centre wells. The gas space contained air. The shaking rate was 90 c.p.m. with a 4 cm. stroke, and the temperature was 20° C.

0·07 vol. of 100% (w/v) trichloroacetic acid (TCA) was added to the contents of the manometer flasks and their washings. This was left at 2° C. for 10 min. with intermittent stirring and then centrifuged at 2700 g. for $712$ min. The supernatant was poured off and the precipitate re-suspended in 2 ml. 7% TCA. This was left at 2° C. with intermittent stirring for 10 min., after which the centrifugation procedure was repeated. 4 ml. ethanol was added to the last centrifugate with thorough mixing, and left for 10 min. at room temperature with intermittent stirring. This was centrifuged at 2700 g. for 5 min. and the supernatant collected. The centrifugate was re-suspended in 2 ml. ethanol and left for 10 min. with intermittent stirring. It was then centrifuged as before and the second supernatant was added to the first. 3 ml. of 3:1 ethanol-ether mixture was added to the last centrifugate, which was re-suspended and placed in a water-bath at 62° C. for 2 min. with stirring. The mixture was centrifuged as before and the supernatant was added to the previous two. These were evaporated at 35-45 mm. Hg in a stream of Ng, the temperature not exceeding 45° C. The dry residue was extracted three times with 3 ml. boiling chloroform, some chloroform-insoluble residue being left behind. The chloroform extracts were evaporated to dryness in a boiling water-bath in a stream of air. The lipid residue was combusted in 0·5 ml. 50% H₂SO₄ with two to three drops of 100 vol. H₂O₂. 2 ml. distilled H₂O was added to the solutions which were then heated for 7 min. in a boiling water-bath. After cooling, the solutions were neutralized to litmus and made up to 10 ml. with distilled H₂O. P determinations were made on 5 ml. aliquots by the method of Fiske & Subbarow (1925). The replicates did not vary by more than 5 %.

The results of one experiment, together with certain relevant calculations, are given in Table 1. The figures involving the dry weight of the 2·67 × 10⁹ spermatozoa in the suspension, i.e. (3) and (11) in Table 1, require explanation. The volume of a sea-urchin spermatozoon is about 18 μ^a (Rothschild, 1950). 1 ml. of this semen (dry weight 139 mg.), which contained 1·78 × 10¹⁰ spermatozoa, therefore consisted of 0-32 ml. spermatozoa and 0·68 ml. seminal plasma. The dry weight of the latter was 39 mg. making the dry weight of the spermatozoa 113 mg. and of 2·67 × 10⁹ spermatozoa 17 mg. Two assumptions are made in the calculations: first, that the phosphorus content of phospholipid is 4% (Maclean & Maclean, 1927), and secondly, that the combustion of 1 mg. phos-pholipid involves the disappearance of 1·6 ml. O₂. The complete combustion of 1 mg. carbohydrate is associated with the disappearance of 0·75 ml. O₂.

The results of all experiments are summarized in Table 2.

Post-chromated Helly-fixed preparations of fresh and aged spermatozoa were examined unstained and after staining with Sudan Black. No differences in structure or lipid concentration were evident on visual examination, but the greatest concentration of Sudan Black stainable material was present in the middle-piece.

When very dilute sperm suspensions (10⁵-10⁶/ml.) were aged for 7 hr. and then fixed in formalin (final concentration 2%), marked differences between the middle-pieces of fresh and aged spermatozoa were seen both with the light and the electron microscope.^* The main differences consisted of an elongation of the middle-piece and a greater separation of it from the head of the spermatozoon.

In spite of the earlier experiments on the carbohydrate content of the spermatozoa and seminal plasma of these sea-urchins (Rothschild & Mann, 1950), we re-estimated the amount of glycogen-like material in a sample of fresh semen, and in an aliquot of the same sample after aerobic incubation, using the same methods as before. 0·15 ml. of fresh semen, sperm density 1·90 × 10¹⁰/ml., contained 0·2 mg. glycogen-like material. After aerobic incubation in the presence of KOH for 390 min. at 20° C., the semen being diluted 1:20 with sea water, the amount of glycogen-like material had fallen to 0·18 mg., a change of about 10%. The complete combustion of 0·02 mg. of this material would involve the disap-pearance of 17 μl. O₂, whereas the observed consumption of oxygen by the same suspension was 361 μl.

Spikes (1948, 1949) found that the total reducing value of a sperm suspension of Strongylocentrotus purpuratus fell by 21·3 mg./g. dry weight of semen during 12 hr. ageing at 19° C. The dry weight of 10⁸ spermatozoa of this species was 0·35 mg., from which, omitting the small weight contribution of the seminal plasma, it follows that i g. of the dried material contained 2-86 × 10¹¹ spermatozoa. Spikes says that 10⁸ spermatozoa consumed some 6μl. O₂ per hour during periods of the order of 12 hr. 2·86 × 10¹¹ spermatozoa would therefore consume about 2X 10⁶μl. O₂ in 12 hr. The disappearance of 2X 10⁶μl. O₂ requires the oxidation of 268 mg. glucose, more than ten times the observed amount. The conclusion is inescapable that Spikes’ experiments do not support his view that the oxidation of intracellular carbohydrate accounts for the oxygen uptake of sea-urchin spermatozoa.

Rothschild & Mann (1950) found 40 mg. glucose/100 ml. of the fresh sperma-tozoa of Echinus esculentus, after hydrolysis with KOH and ethanol precipitation. This means that 2·67 × 10⁹ spermatozoa, the number used in the experiment described in Table 1, contained 1·92 x10⁻² mg. glucose, the oxidation of which would cause the disappearance of 14μ1. O₂, as compared with the observed uptake of the suspension, 448 μl. O₂. Even if it is assumed, as in the calculations relating to Spikes’ experiments, that all the carbohydrate in the semen of E. esculentus is available for oxidation, a very improbable assumption, the observed oxygen uptake cannot be accounted for by carbohydrate oxidation. Sea-urchin semen consists of spermatozoa containing 110 mg. % sugar and seminal plasma containing 200 mg. % of an anthrone-reactive carbohydrate. In the experiment in Table 1, 1 ml. semen contained o-68 ml. seminal plasma and 0·32 ml. spermatozoa. The experimental suspension, which was 3 ml. of semen diluted 1:20 with sea water, contained 0·26 mg. carbohydrate, the oxidation of which would cause the disappearance of some 200 μl. O₂, a little less than half the observed amount. The conclusions from these calcula-tions are consistent with the experiment on glycogen metabolism done this year.

Although a number of substances exert a protective effect on sea-urchin spermatozoa when added to the external medium (Rothschild, 1951), there is no evidence that any of them are utilized to a significant extent as substrates, even though some are carbohydrates.

The capacity of sea-urchin spermatozoa to utilize endogenous phospholipid as a source of energy reveals an interesting and previously unnoticed similarity between these spermatozoa and those of mammals. Normally, the latter obtain the energy necessary for movement by the breakdown of the exogenous substrate fructose, secreted into the seminal plasma by the seminal vesicles (Mann, 1949; Mann & Parsons, 1950). Fructolysis is an anaerobic process; consequently, mammalian spermatozoa are capable of sustained movement in the absence of oxygen. But if suspended in a substrate-free medium, they cannot move unless oxygen is available (Lardy & Phillips, 1941). The same applies to sea-urchin spermatozoa (Harvey, 1930), though in this case the external medium, seminal plasma diluted with sea water, differs from that of mammalian spermatozoa in containing no obvious substrate. Lardy & Phillips also showed that bull spermatozoa suspended in sugar-free Ringer phosphate were able to move in the presence of oxygen, and that in these circumstances there was a decrease in their phospholipid content during incubation. One of the main differences between mammalian and sea-urchin semen is therefore that the former normally contains a source of energy outside the spermatozoa which they can use anaerobically, while the latter does not. This does not imply that sea-urchin spermatozoa are devoid of glycolytic enzymes ; if, however, they are present, their effectiveness is limited by the lack of utilizable substrate in the external medium and the negligible quantity of carbohydrate inside the spermatozoa.

One of us (R.) is indebted to the Medical Research Council for the provision of a laboratory assistant.