1. The respiratory quotient of sea-urchin spermatozoa has been determined with Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus.

  2. The R.Q. of sea-urchin spermatozoa, measured by the Warburg direct method, has been reported to be near unity. This was also the case with the present material when the suspending medium was sea water, the R.Q. being 0·8–1·0. It was found, however, that the pH of sperm suspensions was markedly different in the presence and absence of alkali to absorb CO2.

  3. When the pH of the suspension was fixed by such buffers as 0·025 M-glycyl glycine, the R.Q. measured by the above method was about 0·7. This is in accord with the results of earlier metabolic studies, which indicated that endogenous phospholipids are the main substrates for the respiration of sea-urchin spermatozoa.

  4. The O2 uptake of the present material, however, was found to be little affected by variation in pH. The difference in the R.Q. values obtained with ordinary sea water and buffered sea water, therefore, cannot be explained in terms of pH.

  5. When the spermatozoa were suspended in ordinary sea water, the utilization of endogenous phospholipids was much reduced in the absence of alkali, while in buffered sea water the change in phospholipids was almost the same, with and without the absorption of CO2.

  6. Determination of the R.Q. by the first method of Dickens & Šimer, in which the O2 uptake and the CO2 output were measured with one and the same sperm suspension, gave a value of about 0·7 with both ordinary and buffered sea water.

Sea-urchin spermatozoa are spawned into sea water which contains no obvious nutrient, and accordingly the energy for their movement must be secured exclusively from intracellular reserves. Up to the present time, several investigations have been made on the metabolism of sea-urchin spermatozoa and now there seems to be little doubt that they utilize the oxidation of endogenous phospholipids as the main source of energy for motility (Rothschild & Cleland, 1952; Mohri, 1957a, 1959a). The endogenous phospholipids are first split into fatty acids and glycero-phosphorylesters by the action of phospholipase(s), the liberated fatty acids being then utilized as substrates for aerobic metabolism (Mohri, 1957b, 1959a, c). These facts lead us to expect that the respiratory quotient (R.Q.) of sea-urchin spermatozoa will be approximately 0·7. The R.Q. measurements so far made, however, have rather been near unity (Barron & Goldinger, 1941 ; Hayashi, 1946; Spikes, 1949; Mohri, 1956 *). These measurements of R.Q. were made by the Warburg direct method, which is based on the assumption that the rate of respiration is not affected by the presence or absence of CO2. This assumption, however, has not yet been shown to be justified in sea-urchin spermatozoa and, furthermore, it is known that with certain tissues the presence of CO2 influences the O2 uptake. Rothschild (1956a, b) recently reported that the respiration of sea-urchin spermatozoa is very sensitive to changes in the pH of the suspending medium. According to his results, a change of 0·26 units from pH 7·91 to 8·17 caused an almost twofold increase in O2 uptake. Since sea water possesses a low buffering capacity, it is likely that there exists a considerable difference in pH between sperm suspensions with and without absorption of CO2 if sea water is used as the suspending medium. This would cause a serious error in the measurement of R.Q. by the Warburg direct method. In the present experiments, therefore, these points were examined and an effort was made to obtain a more accurate value of R.Q. for sea-urchin spermatozoa.

The spermatozoa of Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Anthocidaris crassispina, in which the aerobic utilization of endogenous phospholipids has been observed, were employed as material. Semen was obtained as described by Moriwaki (1958) and sperm suspensions were made by diluting semen with sea water, or an appropriate buffer prepared in bicarbonate-free sea water.*

O2 uptake and CO2 output were measured in manometers of the constant-volume type, the gas phase containing air. The R.Q. was determined by two different methods, the Warburg direct method and the first method of Dickens & Šimer (1930). In the Warburg direct method, three flasks of the conventional type were used in order to make corrections for the CO2 retention in the medium. Two ml. of sperm suspension was placed in each flask. The O2 uptake of the suspension during the experimental period was known from flask 1 which contained 0·2 ml. of 10% KOH in the centre well In flasks 2 and 3, 0·2 ml. of SN-H2SO4 was placed in the side-arm and tipped at the end and at the start of the experimental period, respectively. The CO2 output was then calculated from the following equation
where h2 and h3 represent the final manometer readings of flasks 2 and 3 after the addition of acid from the side-arm. is the O2 constant of flask 2. and refer to the CO2 constant of flasks 2 and 3. The experimental period was 60 min., and temperature was 20° C. with Pseudocentrotus and 28° C. with Anthocidaris.

In the first method of Dickens & Šimer, the O2 uptake was first determined by absorbing the evolved CO2 in alkali, and then all the CO2 was liberated by adding acid to both sperm suspension and alkali at the end of the experimental period. The measured CO2 must be the sum of the initial CO2 and the CO2 evolved during the experimental period. To obtain the initial CO2 another manometer was used, in which acid was added at the start of the experimental period. The flasks used were of the special type designed by Dickens & Šimer (1930), having a trough encircling the main vessel and a side-arm. Two ml. of sperm suspension was placed in the main vessel 0 5 ml. of cold saturated Ba(OH)2 was put in the trough and 0· 5 ml. of 3 N-H2S04 was placed in the side-arm. The experimental period was 30 min. with Pseudocentrotus and 120 min. with Hemicentrotus. Temperature was 20° C.

The phospholipid content was determined in the manner described by Mohri (1959a). Acid-hydrolysable carbohydrate was determined by measuring the reducing sugar according to the method of Nelson (1944) after hydrolysis of the tissue with 1 N-HCl for 5 hr. and precipitation of proteins with a mixture of ZnSO4 and Ba(OH)2.

The pH of the medium was measured by glass electrodes. When buffer was used, the pH of the buffer solution was adjusted to that of sea water, about 8·2 at Misaki. The sperm density was determined with a haemocytometer.

R.Q. measured by Warburg direct method. As mentioned in the Introduction, previous studies, which gave an R.Q. of sea-urchin spermatozoa near unity, were made by the Warburg direct method, without sufficient control of the pH by means of a suitable buffer. In this work, therefore, the R.Q. was first measured by the same method, with both buffered and unbuffered medium. The results summarized in Table 1 indicate that the R.Q. obtained with ordinary sea water as diluent was somewhat lower than, but not very different from, the values hitherto reported—about 0·9. On the other hand, when sea water was buffered with either 0·025 M-glycyl glycine or 0·015M-veronal the R.Q. was about 0·7, as would be expected from the results of previous chemical analyses. The question then arose as to why such a discrepancy was observed.

Table. 1.

Respiratory quotient of sea-urchin spermatozoa measured by the Warburg direct method. Sperm density, 3·1-8·8 × 108spermatozoa/ml.

Respiratory quotient of sea-urchin spermatozoa measured by the Warburg direct method. Sperm density, 3·1-8·8 × 108spermatozoa/ml.
Respiratory quotient of sea-urchin spermatozoa measured by the Warburg direct method. Sperm density, 3·1-8·8 × 108spermatozoa/ml.

Changes in pH of sperm suspensions during manometric experiments

Fig. 1 shows the changes in the pH of sperm suspensions during the manometric procedure with and without the absorption of CO2 by means of KOH in the centre well. Three flasks with the same contents were used to obtain each curve. Spermatozoa were added to the suspending medium at t = o and the pH was determined. After 15 min. for temperature equilibration, the manometric readings were taken during the following 60 min., the pH being checked at the start and the end of the experimental period. When ordinary sea water was used, there occurred a sudden drop of pH from the original value, 8·20 in this case, to 7·92 immediately after the addition of spermatozoa. In the flask with KOH, however, the change in the pH of the suspension was slight thereafter, while in the flask without KOH the pH continued to drop during the equilibration period. In consequence, the respiration proceeded at different pH’s during the experimental period in the flasks with and without KOH, at about 7·8 and 7·3, respectively. On the other hand, when sea water was buffered with 0·025 M-glycyl glycine, little change was observed in the pH of suspension in either case, the pH remaining not far from the original value.

Fig. 1.

Changes in pH of sperm suspensions of the sea-urchin, Pseudocentrotut dept es tut, during manometric experiments with (solid circle) and without (open circle) absorption of CO2 by KOH. Dotted line indicates pH of ordinary sea water. Suspending medium : lower two curves, sea water; upper two curves, 0·025 M-gly. gly.-sea water. Sperm density, 5·2 × 108/ml. Temp., 20° C.

Fig. 1.

Changes in pH of sperm suspensions of the sea-urchin, Pseudocentrotut dept es tut, during manometric experiments with (solid circle) and without (open circle) absorption of CO2 by KOH. Dotted line indicates pH of ordinary sea water. Suspending medium : lower two curves, sea water; upper two curves, 0·025 M-gly. gly.-sea water. Sperm density, 5·2 × 108/ml. Temp., 20° C.

As already mentioned, Rothschild (1956b) reported that the O2 uptake of sea-urchin spermatozoa declines with a decrease in the pH of medium, particularly on the acid side of pH 8. If the same is true in the present material, the O2 uptake would be less in the flask without KOH than in the flask with KOH in the case of ordinary sea water as diluent. Assuming that the metabolic sequence is unaffected by the presence and absence of CO2, i.e. the R.Q. is the same under both conditions, , in equation (1) calculated from the obtained data would give a smaller negative value (h2 < h3 in the usual case) as compared with the value expected from the fact that there is no difference in the O2 uptake between the two flasks. Consequently, the amount of calculated from equation (1) would become larger than the true value, resulting in a higher R.Q. value. The use of buffered sea water would exclude such errors. As will be described below, however, the problem was not solved merely by considering the difference in the pH.

Effect of pH on O2 uptake of sperm suspension

To examine the problem of whether the O2 uptake of the present material shows a marked change with variation in the pH, an experiment was performed under the same conditions as those used by Rothschild (1956b). Sperm suspensions were prepared with 0·05M-gly. gly.-sea water having different pH’s, and the O2 uptake was measured during 60 min. The results are shown in Fig. 2, in which each point indicates the average value of the pH before and after the experiments, the range being indicated by the horizontal line. In contradiction to the results obtained with Echinus esculentus and Paracentrotus Uvidus, the O2 uptake of the present material was little affected by the change in the pH, although there was a tendency for the rate of O2 uptake to decrease towards both extremes of the range used. Identical experiments using tris(hydroxymethyl)aminomethane-sea water and borate-sea water gave similar results in all cases. At least in the present material, therefore, the difference between the R.Q.’S obtained with ordinary sea water and buffered sea water could not be explained by a variation in the O2 uptake due to the change in the pH of suspending medium, as postulated above.

Fig. 2.

Effect of pH on O2 uptake of spermatozoa of the sea-urchin, Pieudocentrotus deprasut. pH of sperm suspensions was fixed with 0·05 M-glycyl glycine. Sperm density, 4·5 x 108/ml. Temp, 20° C.

Fig. 2.

Effect of pH on O2 uptake of spermatozoa of the sea-urchin, Pieudocentrotus deprasut. pH of sperm suspensions was fixed with 0·05 M-glycyl glycine. Sperm density, 4·5 x 108/ml. Temp, 20° C.

In the absence of KOH, almost all the respiratory CO2 is chemically bound in a buffer such as glycyl glycine at high pH values. According to a calculation made by Rothschild (personal communication), using a manometer with a constant of 2 and 2 ml. fluid, 90 % of the evolved CO2will be retained in 0·025 M-glycyl glycine, pH 8·2, at 15° C. It is necessary to ascertain whether such a situation does not have an influence on the metabolism of sea-urchin spermatozoa.

Effects of presence and absence of CO2 on changes in phospholipid content of spermatozoa

In the course of studies on phospholipid metabolism of sea-urchin spermatozoa it has been noticed that diminution of phospholipids during aerobic incubation is very small when the respiratory CO2 is not removed with KOH (Mohri, 1959a). As presented in Table 2, this was confirmed in the present experiments. When sperm cells were suspended in ordinary sea water, the phospholipid content diminished to about half the initial value (2·18–2·46 mg./1010 spermatozoa) during a 6 hr. incubation at 20° C. in the presence of KOH, whereas only about one-sixth of the phospholipids disappeared during the same incubation period in the absence of KOH. Even if 0·025 M-gly. Gly.-sea water was used instead of ordinary sea water, the change in the phospholipid content was influenced by the presence or absence of KOH. In this case, however, the difference was not as marked as that observed in ordinary sea water. The difference may be less during the experimental period used in the R.Q. measurements (the first 60 min.). Under the conditions which inhibited phospholipid utilization, compensatory breakdown of carbohydrates could not be observed. The content of acid-hydrolysable carbohydrate changed, for instance, from 0·35 to 0·30 or 0·31 mg./1010 spermatozoa during 6 hr. incubation at 20° C. in all cases.

Table. 2.

Respiratory quotient of sea-urchin spermatozoa measured by the Warburg direct method. Sperm density, 3·1-8·8 × 108spermatozoa/ml.

Respiratory quotient of sea-urchin spermatozoa measured by the Warburg direct method. Sperm density, 3·1-8·8 × 108spermatozoa/ml.
Respiratory quotient of sea-urchin spermatozoa measured by the Warburg direct method. Sperm density, 3·1-8·8 × 108spermatozoa/ml.

Thus, assuming that the respiration of sea-urchin spermatozoa is almost entirely due to the oxidation of endogenous phospholipids, the rate of O2 uptake would be lower in the absence than in the presence of KOH, especially when ordinary sea water is used as the medium. This would explain at least in part the above-observed discrepancy between the values for R.Q. with ordinary sea water and with buffered sea water.

R.Q. measured by the first method of Dickens & Šimer

In the determination of the R.Q., the first method of Dickens & Šimer is more favourable than the Warburg direct method, since the measurements of O2 and CO2 are both carried out on the same sperm suspension, thus excluding possible errors due to the use of two suspensions under different conditions, which is inevitable in the Warburg direct method. Although the CO2 initially present is measured with another sperm suspension, the difference between any two suspensions is considered to be negligible. As shown in Table 3, the R.Q. obtained by this method was about 0·7 with either ordinary sea water or 0·025 M-gly. gly.-sea water. The duration of the experiment was far longer with Heimcentrotus than with Pseudocentrotus (120 and 30 min., respectively), but the results obtained were identical.

It has been reported that the initial high respiration following dilution of semen with sea water is sensitive to the action of sodium azide, while the subsequent steady low respiration is little affected by this agent. Conversely, 2,4-dinitrophenol or substrates such as lecithin and octanoate do not affect the initial respiration, but stimulate the rate subsequently (Mohri, 1956, 1957a). An experiment, undertaken to examine whether different substrates are used in the initial high and the steady low respiration, resulted in a similar R.Q. in the two cases; for example, the R.Q. measured during the first 15 min. after dilution was 0·73, while that during the 90 min. after incubation for 180 min. at 20° C.was 0·71.

The concept that the endogenous phospholipids provide the energy for maintenance of motility of sperm cells was first put forward by Lardy & Phillips (1941a, b) in washed bull spermatozoa under aerobic conditions. Evidence in favour of this concept, however, has not been obtained in later experiments with washed boar and bull spermatozoa (Ogura, 1955 ; Bomstein & Steberl, 1957), in which neither a considerable decrease in the content of lipid phosphorus during aerobic storage nor oxidation of exogenous phospholipid by sperm cells could be demonstrated. Quite recently, the concept has been revived with some modifications through the work of Mann and his colleagues (Lovern, Olley, Hartree & Mann, 1957; Hartree & Mann, 1959), who found that the main component of phospholipids in ram and bull spermatozoa is not ester phospholipid but a choline-containing plasmalogen. Liberation of fatty acid from the plasmalogen necessitates a reduction in the ester linkage, but not a change in the content of lipid phosphorus. These authors measured the R.Q. of washed ram spermatozoa by the Warburg indirect method and obtained a value between 0·70 and 0·72. Thus, most of the substrates for the endogenous respiration of washed mammalian spermatozoa seem to consist of the fatty acids derived from phospholipids.

In sea-urchin spermatozoa, which when shed into sea water are in a state resembling that of washed mammalian spermatozoa, the data hitherto obtained are consistent with the above concept, except that the early determinations of the R.Q. produced a value suggesting carbohydrate metabolism. The phospholipids of sea-urchin spermatozoa, however, mainly consist of ester phospholipids, plasmalogen making up only a small portion (Hartree & Mann, 1959; Mohri, 1959a,b). The results presented above indicate that the R.Q. of about unity obtained by the Warburg direct method in ordinary sea water is a misleading one, owing to the metabolic difference between sperm suspensions with and without the absorption of respiratory CO2. In fact, in ordinary sea water the endogenous phospholipids were metabolized at a much reduced rate in the absence, as compared with in the presence, of KOH. As there is no compensating fall in sperm carbohydrate content, O2 uptake should fall. Judging from the results obtained in the present experiments, the cause underlying such a difference is probably the evolved CO2 or the bicarbonate content of the medium, although a dramatic change in the O2 uptake accompanying variation in pH was reported in other sea-urchin species by Rothschild (1956b).

In connexion with the respiratory dilution effect, which is defined as the increase in O2 uptake per spermatozoon in dilute sea-urchin sperm suspensions as compared with dense ones (cf. Rothschild, 19566), Runnstrom, Tiselius & Lindvall (1945) presented the hypothesis that the CO2 content of dense suspensions is probably the factor which inhibits sperm motility. This hypothesis has not been taken into consideration, because in the manometric experiments in which the respiratory dilution effect is observed, the evolved CO2 is constantly removed by KOH (Rothschild, 1948). The present results, however, seem to agree with this old hypothesis, although direct evidence is still lacking.

On the other hand, the R.Q. values, measured either by the Warburg direct method in buffered sea water or by the first method of Dickens & Šimer, were close to 0·7, a value consistent with the results of early studies. In buffered sea water, there was no marked difference in the utilization of endogenous phospholipids with and without the absorption of CO2. Strictly speaking, these measurements, especially the latter, were performed under rather special conditions, in which almost all the CO2 was removed and the bicarbonate content of medium also reduced, being thus somewhat different from the natural circumstances. The experiments in which the utilization of phospholipids was clearly demonstrated were also done under such conditions (Rothschild & Cleland, 1952; Mohri, 1957a, 1959a). Although measurements using the indirect method are desirable in this respect, it cannot be said that the experimental conditions in this method are the same as the natural ones.

We wish to thank Prof. J. Ishida of Tokyo University and Dr J. C. Dan of Ochano-mizu Women’s University for their valuable criticisms and kind help in preparing the manuscript. We also thank Lord Rothschild of Cambridge University for his kind suggestions. We are much indebted to the Director and Staff of the Misaki Marine Biological Station for their kindness and hospitality during the course of this work.

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*

In this paper the explanation of Fig. 4 (p. 78) should be corrected as follows: (1)→(3); (2)→(1); (3)→(2).

*

Abbreviations such as 0·025 M-gly. gly-sea water, which means sea water buffered with 0·025 M-glycyl glycine, will be used in the present paper.