ABSTRACT
The effect of on the respiration and motility of sea-urchin spermatozoa was studied on Anthoddaris crassispina. Some points were also corroborated on Hemicentrotus pulcherrimus, Pseudocentrotus depressus, Paracentrotus lividus and Sphaerechinus granularis.
It was found that any level of CO2 above 1 %, both in oxygen and in air, inhibited the O2 uptake of spermatozoa suspended in sea water, measured polarographically with a vibrating platinum electrode. The inhibitory effect paralleled the and was completely reversed by introducing oxygen or air.
pH variations between 8·50 and 6·75 had no influence on O2 uptake, when the pH was stabilized with 0·05 Mhistidine-HCl-NaOH. O2 uptake was, however, reduced to some extent outside this range, especially on the acid side. Although the increase in is inevitably followed by a decrease in pH, the inhibitory effect of CO2 far exceeds that caused by the reduction in pH.
The O2 uptake rate was little affected by the addition of both bicarbonate and carbonate ions to the suspending medium, although the former had a slightly stimulating effect at certain concentrations.
In buffered sea water, CO2 had little influence on O2 uptake even at partial pressures as high as 10% which inhibited the bulk of O2 uptake in sea water.
Sperm motility was also inhibited by CO2. In this case, too, the inhibition paralleled the and was completely reversible. The effect was more pronounced in air than in oxygen, and in dense sperm suspensions than in dilute ones.
These results suggest that gaseous CO2 is the factor responsible for the inhibitory effect. The possible role of CO2 in the dilution phenomena of sea-urchin spermatozoa is discussed.
INTRODUCTION
It has long been known that sea-urchin spermatozoa remain immotile in the testis or semen and are activated only following shedding into sea water or dilution with seawater. Up to the present, many efforts have been made to clarify the nature and the causes of such dilution phenomena or of the initiation of movement and the following factors have been reported to be responsible, although some of them should be excluded in view of present knowledge (cf. Rothschild, 1951, 1956a; Bishop, 1962):
(1) CO2narcosis
Cohn (1918) considered the narcotic effect of CO2, low O2 concentration and low pH as the environmental conditions responsible for the lack of movement in semen of Arbacia punctulata, and that dilution with sea water reversed these conditions with consequent activation of the spermatozoa. According to Gray (1928), however, the explanation in terms of CO2 ‘is only partially correct at the best, and quite erroneous’ (p. 342), because dilute sperm suspensions of Echinus esculentus and Psammechinus miliaris respire at a higher rate, per unit number of spermatozoa, than dense ones, when the O2 uptake is measured by the Warburg direct method which necessitates the continuous removal of respiratory CO2. Gray’s finding has repeatedly been confirmed by many other workers with several sea-urchin species (Rothschild, 1948b,1950, 1956a; Rothschild & Tuft, 1950; Rothschild & Tyler, 1954; Barron, Gasvoda & Flood, 1949; Mohri, 1956a), and has been called Dilution Effect, Dilution Effect II or Respiratory Dilution Effect. He also reported that dilute suspensions consume more oxygen in active life than dense ones (Dilution Effect I). Since then, CO2 narcosis has long been left out of consideration from the abovementioned reasoning (cf. Rothschild, 1948b), although Runnstrôm, Tiselius & Lindvall (1945) once suggested that CO2 content of dense suspensions probably was a sufficient inhibitor of sperm motility in Strongylocentrotus droebachiensis and Echinocardium cordatum. Recently, however, the effect of CO2 has again been taken into consideration in relation to dilution phenomena both by Rothschild (1956a, b) and by Mohri & Horiuchi (1961) from somewhat different standpoints as will be seen below.
(2) O2tension
Rothschild (1948a) suggested that E. esculentus spermatozoa do not move in undiluted semen because of the lack of O2. The O2 tension in the semen was less than one-tenth of that in air, and the spermatozoa were able to move even in undiluted semen if placed in a gas chamber filled with pure O2. The fact that sea-urchin spermatozoa are unable to move under anaerobic conditions has been reported by other workers (Harvey, 1930; Barron, 1932). Furthermore, the O2 uptake of dense sperm suspensions was altered by changes in O2 tension, but that of dilute suspensions was not affected by varying . This was explained by Rothschild (1948 b) as the inability of a significant fraction of the spermatozoa to respire in dense suspensions through lack of O2. In the same paper, he described how CO, a potent inhibitor of cytochrome oxidase, has a greater inhibitory action on dilute than on concentrated sperm suspensions. Mohri (1956b) also observed a similar action of sodium azide, another inhibitor of cytochrome oxidase. In a following paper, Rothschild (1950) showed that Dilution Effect I, the increased total O2 uptake of dilute suspensions, is observed only when the O2 saturation in dense suspensions during manometric experiments is not satisfactory. On the other hand, if this condition is satisfactory, rather dense suspensions consume more O2 during their active life. The problem of O2 saturation, therefore, must always be borne in mind. Since Dilution Effect II or the Respiratory Dilution Effect occurs even when the O2 saturation seems to be satisfactory (Rothschild & Tuft, 1950), some other factors than O2 tension must be involved in this effect. At any rate it seems certain that O2 tension is an important factor in inducing sperm motility after dilution of semen with sea water.
(3) Allelostasis
This idea was presented by Gray (1928) in his paper dealing with the first quantitative analysis of Dilution Effect. He concluded that the activating effect of sea water must be mechanical, the spermatozoa becoming surrounded by an increased free space for movement on dilution. In other words, spermatozoa have an allelostatic effect on the activity of their neighbours in undiluted semen or in dense suspensions. According to Rothschild (personal communication) the effect is explained more precisely as follows. In a dense suspension the velocity fields of different spermatozoa will interfere with each other so that any particular spermatozoon will be in a medium with a higher viscosity than that of the medium without spermatozoa. The activity of spermatozoa is reduced in media of unusually high viscosity. As described above, however, spermatozoa can move in undiluted semen provided the O2 supply is sufficient. It is difficult, therefore, to explain the cause of the Dilution Effect merely in terms of allelostasis, although, when activated by O2, spermatozoa in semen do not move so vigorously as after dilution (Rothschild, 1948b).
(4) Kions
A reduction in K ions through dilution was regarded by Schlenk & Kahmann (1938) as playing a significant role in initiating motility in rainbow trout spermatozoa. In fact, the concentration of K ions in seminal plasma of E. esculentus is about four times as high as in sea water (Rothschild, 1948 a). Since, however, dilution with seminal plasma instead of sea water has no adverse effect on sperm activity (Gray, 1928; Hayashi, 1945, 1946; Rothschild, 1948a, b), this factor could be excluded from the cause of the Dilution Effect in sea-urchin spermatozoa, although it has been reported that spermatozoa are completely immotile in isotonic (0·5 M) KC1 solution (Moriwaki, 1958).
(5) Androgamone I
This hypothesis was put forward by German workers (Hartmann, Schartau & Wallenfels, 1940). They obtained a factor called Androgamone I by methanol extraction of dried sperm of Arbacia pustulosa, and ascribed the lack of movement in semen and the senescence of spermatozoa after dilution to this substance. They believed that the substance is present in seminal plasma. The existence of such a substance in seminal plasma obtained after centrifugation of semen was reported by Southwick (1939) with Echinometra subangularis. The role of Androgamone I in sea-urchin semen was soon questioned by Runnström et al. (1945), although they proved that this factor is responsible for the lack of movement in undiluted salmon semen. The foregoing fact that no special inhibitory substances are contained in seminal plasma also denies the proposed role of Androgamone I. This hypothesis, therefore, should be abandoned in view of the present knowledge.
(6) Trace metals, Cu and Zn
Rothschild & Tuft (1950) showed that Dilution Effect occurs when small amounts of Cu or Zn ions are added to a dense sperm suspension. In dilute suspension, however, there is no Dilution Effect when these ions are added. The effect of these ions is counteracted by adding diethyldithiocarbamate, a chelating agent. From these facts, they came to the conclusion that Dilution Effect is at least in part caused by these metals, particularly Cu, in sea water. Mohri (1956a) and Utida & Nanao (1956a) also confirmed the stimulating action of Zn ions on O2 uptake of dense suspensions in Pseudocentrotus depressus and Hemicentrotus pulcherrimus. It has been reported, on the other hand, that both the addition of chelating agents such as glycine (Tyler & Rothschild, 1951) or versene (Rothschild & Tyler, 1954) and the previous removal of heavy metals from sea water by shaking with dithizone solution (Utida & Nanao, 1956a) nullify the Dilution Effect. Furthermore, Mizuno (1956) indicated that sea-urchin spermatozoa show a tendency to take up Zn ions from the surrounding medium during incubation.
As a possible mechanism by which trace metals exert their Dilution Effect, Mohri (1956b) tentatively suggested that the reaction rate of the cytochrome-cytochrome oxidase system is increased by adding these metals as a result of the masking of soluble SH groups which are believed to regulate the oxidation rate of cytochrome c (Barron, Nelson & Ardao, 1948). In fact, the cytochrome-cytochrome oxidase system attains its full action only in dilute suspensions (Mohri, 1956b; cf. Rothschild, 1948b). Rothschild (1956a), on the other hand, proposed that trace metals cause uncoupling of oxidation and phosphorylation which he considered as being responsible for the Dilution Effect. This assumption is based on the fact that 2,4-dinitrophenol (DNP) stimulates the O2 uptake of dense suspensions more than dilute ones, and that the effect of DNP is reduced in the presence of versene. Similar results were obtained by Mohri (1956a) with DNP and by Nelson (1948) with usnic acid, another uncoupler of oxidation and phosphorylation. It is known that DNP enhances the adenosinetriphosphate (ATP) dephosphorylating activity in mitochondria, thus bringing about a reduction in P/O ratio. It is still uncertain whether ATPase in sperm mid-piece, i.e. in mitochondria, of sea-urchin spermatozoa is affected by DNP, although ATPsplitting activity of whole spermatozoa is somewhat accelerated by this agent (Mohri, 1958). In connexion with the action of trace metals it is suggestive that ATP-splitting activity of the sperm ‘head’ (head proper plus mid-piece) is activated by Zn ions (Mohri, 1958). On the other hand, extensive studies on the motility-inducing mechanism in starfish spermatozoa have been made by Japanese workers (Fujii, Utida, Mizuno & Nanao, 1955; Mizuno, 1956; Kinoshita, 1956a, b;Utida & Nanao, 1956a, b). They showed that histidine and other chelating agents induce sperm motility in starfish and in some other marine forms and conceived that the release of Zn from spermatozoa is responsible for the sperm activity. This assumption, however, needs further facts to support it, and is not applicable at least to the Dilution Effect in sea-urchin spermatozoa, where the sperm activity is reduced by chelating agents and raised by trace metals. A more recent experiment by Rothschild (1956b) throws some doubts on the role of trace metals in the Respiratory Dilution Effect, because the effect can still be observed when the suspending medium is 0·05 M borate-buffered sea water with 10-3 M versene.
Besides having action on O2 uptake, several amino acids and other chelating agents produce a prolongation of the life-span of sea-urchin spermatozoa (Tyler & Atkinson, 1950; Tyler & Rothschild, 1951; Tyler, 1953; Rothschild & Tyler, 1954; Mohri, 1956b). The results are explained by the removal of trace metals, Cu and Zn, in sea water, which are harmful to the spermatozoa, although they induce Respiratory Dilution Effect described above.
(7) pH
The pH of seminal plasma has been reported to be 7·6−7·9 in A. punctulata (Hayashi, 1945) and 7·5 in E. esculentus (Rothschild, 1948a), both being lower than that of sea water which is about 8·2. As mentioned above, Cohn (1918) suggested that low pH in semen is one of the important factors inhibiting sperm activity. This possibility was once excluded because seminal plasma does not have an adverse effect on sperm activity and spermatozoa are motile at pH’s lower than 7·3 (Rothschild, 1948a). Recently, however, the problem was again taken up by Rothschild (1956a, b), using rather strong buffers such as 0·05 M glycyl-glycine to fix the pH. According to his results, an increase of 0·26 unit from pH 7·91 to 8·17 is associated with an almost twofold increase in O2 uptake of E. esculentus spermatozoa. As sea water has only poor buffering capacity, it is possible that such a difference in pH between dense and dilute sperm suspensions when sea water is used as dilution medium, is due to the presence of more CO2 in the dense ones even with constant removal of the gas by KOH. He makes the following statement (1956a, p. 170),’the Respiratory Dilution Effect previously observed after addition of sea water to dense suspensions… may therefore have been “artifacts”, due to the increased sperm activity at high pH’s’. However, pH does also play some role in general dilution phenomena, i.e. activation of spermatozoa after dilution, especially in this species. Unfortunately, his conclusion does not hold for all sea-urchin species, because the O2 uptake of several Japanese seaurchins (P. depressus, H. pulcherrimus and Anthocidaris crassispina) is little affected by varying the pH from 7·0 to 8·5 under the same experimental conditions as those used by Rothschild (Mohri & Horiuçhi, 1961). We must expect, therefore, the presence of some other factors than pH essential for the Dilution Effect.
As described above, there are confusing features in the arguments about the cause of the Dilution Effect, and none of the proposed explanations is conclusive, except O2 saturation. Before going further, it is necessary to define exactly what the term ‘Dilution Effect’ means, in order to avoid unnecessary confusion in this respect. In the present paper, Dilution Effect is used for general dilution phenomena, including activation of respiration and motility of sea-urchin spermatozoa following dilution with sea water, and Respiratory Dilution Effect for the increase in O2 uptake per unit number of spermatozoa, in dilute as compared with dense suspensions, when measured by the Warburg direct method, as defined by Rothschild (1956b).
In our previous papers (Mohri, 1959; Mohri & Horiuchi, 1961) it was reported that the utilization of endogenous phospholipids during ageing of sea-urchin spermatozoa is much more reduced in the absence of KOH than in its presence when this substance is used to absorb the respiratory CO2. Since most of the O2 uptake in sea-urchin spermatozoa is accounted for by oxidation of endogenous phospholipids (Rothschild & Cleland, 1952; Mohri, 1957), and since no compensatory utilization of endogenous carbohydrates occurs without absorption of CO2 (Mohri & Horiuchi, 1961), the reduction in phospholipid utilization should be reflected in a fall in O2 uptake. In the present experiments, therefore, the effect of CO2 was examined by following the O2 consumption polarographically, in parallel with observation of sperm motility.
MATERIALS AND METHODS
Most experiments were done with the spermatozoa of a Japanese sea-urchin, Anthocidaris crassispina. Some points were checked with the spermatozoa of other Japanese sea-urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus and of the Mediterranean sea-urchins, Paracentrotus Iividus and Sphaerechinus granularis. The semen was obtained by the KCl-injecting method as described by Moriwaki (1958). One ml. of semen thus obtained contains about 5−6 × 1010 spermatozoa in Japanese sea-urchins and about 4−5 × 1010 spermatozoa in Mediterranean sea-urchins. Sperm suspensions were made by diluting semen with filtered sea water to 1 :100 throughout the present experiment, unless otherwise mentioned. Filtered sea water was boiled and made up to its original volume with distilled water after cooling.
The O2 consumption of a sperm suspension was followed polarographically with a vibrating platinum electrode developed by Chance & Williams (1955). The general scheme of apparatus is presented in Fig. 1. The platinum electrode was vibrated up and down with an amplitude of about 0·5 mm. at 100 cyc./sec. The electrode is’ polarized at—0·6 V. against a calomel reference electrode. For measurements of O2 consumption, 5 ml. of sperm suspension were put into one side of the H-shaped trough of the apparatus and the platinum electrode was immersed in the suspension. Gas mixtures of the desired composition were then introduced with a glass capillary and bubbled through the suspension until the galvanometer reading reached its maximum. About 200 ml. of gas were found to be sufficient for attaining this condition. Readings were taken immediately after the bubbling of the gas mixture was stopped. The trough was covered with a few cover-glasses during the measurement to prevent the diffusion of O2 into air.
Sperm motility was observed under an ordinary microscope. The sample was taken from the sperm suspension in the trough to a hollow slide and covered with a coverglass as quickly as possible with minimum disturbance. The motility was roughly classified into five grades: very vigorous movement (+ + + +), vigorous movement (+ + +), moderate movement ( + + ), faint movement ( + ) and motionless (−). Sometimes, the score ( ± ) was also used in those cases where very few spermatozoa in the microscopic field were motile.
RESULTS
Effect of CO2 tension on O2 consumption
Fig. 2 shows the effects of differing proportions of CO2 in pure O2 on the O2 uptake of sea-urchin spermatozoa as measured polarographically by the decrease in O2 tension of the medium. The results were obtained with a single sperm suspension of A. crassispina. With each gas mixture, readings were taken for 5 min. at 1 min. intervals, and then one gas mixture was replaced by another and so on. Each curve is corrected for the slight decrease in O2 tension which occurred by diffusion in the absence of spermatozoa. It is apparent that a marked inhibition of O2 uptake occurs with increasing tension of CO2. With 1% CO2, the O2 consumption rate was little affected, but never exceeded that with 100% O2. After severe inhibition with 50% CO2, the O2 uptake of the suspension was completely restored to the initial rate by replacing the gas mixture again with 100 % O2. The observed effect of CO2 is not due to the reduction in the initial O2 tension in the sperm suspension caused by the mixture of O2 with CO2, because no reduction in O2 uptake with decreasing initial O2 tension was obtained in a control experiment where CO2 was replaced by N2, although the rate was somewhat altered with concentrations of N2 above 50 %. Similar results were obtained with the spermatozoa of other Japanese sea-urchins.
Fig. 3 shows the results obtained by varying in air. With 100% air, O2 uptake proceeded at much slower rate compared with that with 100 % O2, as was expected from the above result that the reduction of O2 tension below 50 % brought about a slowing down of the O2 consumption rate. Nevertheless, the inhibitory effect of CO2 was apparent. In this case, too, the inhibition was completely reversible. The percentage inhibition with any given concentration of CO2 was almost the same in both O2 and air.
Effect of pH on O2 consumption
The increase in is necessarily accompanied by a reduction in pH, when the suspending medium is sea water. Although it has already been shown that spermatozoa of Japanese species resist the changes in pH between 7-0 and 8-5 (Mohri & Horiuchi, 1961), in contrast with spermatozoa of E. esculentus Which are very sensitive to pH (Rothschild, 19566), actual pH determinations with glass electrodes after measurements of O2 uptake in A. crassispina gave the values 7·1 for 2·0% CO2, 6·8 for 5 % CO2 and 6·1 for 10% CO2. These values are a little higher than the theoretical values for sea water, probably owing to the buffering action of the spermatozoa themselves. The values obtained with P. lividus during the winter season were 6·9 for 2 % CO2, 6·4 for 5 % CO2 and 5·9 for 10 % CO2. It is necessary, therefore, to re-examine the influence of pH on O2 uptake, especially on the acid side beyond 7·0. In these experiments, the pH was controlled with 0·05 M histidine-HCl-NaOH buffer, and 100% O2 was used as the gas phase. The results are shown in Fig. 4, where each curve was obtained with different sperm suspensions from the same semen sample. The O2 uptake of the material was hardly affected by the changes in pH from 8·50 to 6·75, and proceeded at about the same rate as in unbuffered sea water. Outside this range, the O2 uptake gradually declined with both increasing and decreasing hydrogen ion concentration. The magnitude of the reduction due to the decrease in pH, however, was not so large as that obtained by increasing the , as can be seen by comparing the curves in Figs. 2 and 4. For instance, the inhibition of O2 uptake by 5 % CO2 amounts to 57 %, while that following the reduction in pH to 6·5 is only 16 %. Unless histidine buffer has some special effect other than via pH on sperm activity, especially on the acid side, this might be interpreted as showing that the inhibitory effect of CO2 is not due merely to the fall in pH of the suspending medium, at least in the present material.
Effect of bicarbonate and carbonate ions on O2 consumption
An experiment was then undertaken to see whether bicarbonate and carbonate ions have an inhibitory effect on O2 uptake of sea-urchin spermatozoa, using 100% O2. Different suspensions from a semen sample were used at each concentration of bicarbonate and carbonate. As shown in Fig. 5, no inhibition was caused by any concentration of these ions used, bicarbonate showing rather a slight stimulating action between the concentrations of 5 and 20 μM/ml. The results indicate that gaseous CO2 or free CO2 is the possible factor responsible for the observed inhibitory effect.
Effect of buffer on inhibitory action of CO2
It has been reported that when buffered sea water is used, the utilization of endogenous phospholipids proceeds at almost the same rate, with and without the absorption of CO2, in contrast to the marked reduction of phospholipid utilization which occurs without absorption of CO2 when ordinary sea water is the suspending medium (Mohri & Horiuchi, 1961). This suggests a protective action of buffer against the adverse effect exerted by CO2. As the curves in Fig. 6 indicate, in 0·05 M histidine-sea water, pH 7·25, the O2 uptake was only slightly modified by CO2 even at high concentrations such as 10% which has a marked inhibitory effect in ordinary seawater. The results again support the view that free CO2 is necessary for the inhibitory effect, bound CO2 possessing no such effect.
Effect of CO2 tension on sperm motility
In parallel with the measurements of O2 consumption, sperm motility was examined with increasing CO2 tension. Some results obtained with A. crassispina are presented in Table 1. Motility decreased inversely as the concentration of CO2 and the spermatozoa did not move at CO2 tensions above 8 %, when CO2 was mixed with pure O2. In spite of a complete cessation of movement, a small amount of O2 is still taken up at concentrations of CO2 above 10 %, as can be seen from the curves in Fig. 2. Motility was regained by introducing 100% O2 after complete cessation. In the same table the results of observations with 0·05 M histidine-sea water, pH 7·4, are also listed. In the buffered sea water the motility was suppressed a little as compared with that in ordinary sea water. As would be expected from the results of O2 uptake measurements, however, there was no inhibition of movement even after exposure to 10 % CO2, the spermatozoa moving rather more vigorously than in 100% O2.
Tables 2 and 3 summarize the results obtained with the spermatozoa of P. lividus. When the gas phase was O2, the sperm motility was suppressed completely by about 8 % CO2, as in the case of A. crassispina. When CO2 was mixed with air, on the other hand, only 3 % was sufficient to stop the movement. The same was true in spermatozoa of other species. This might be due to the fact that there exists a minimum amount of O2 uptake necessary for maintenance of motility, and that the O2 uptake is more easily reduced to this level by increasing the tension of CO2 in air than in pure O2, since the O2 uptake rate is smaller in the former than in the latter, as described above. Another alternative is that O2 has a protecting action of some kind against the inhibitory effect exerted by CO2. The effects of on motility in sperm suspensions of different densities are shown in Table 3. It is apparent that more concentrated suspensions are more susceptible to the increasing tension of CO2. In fact, with 2 % CO2 in O2 the spermatozoa appeared to move more vigorously in a suspension of 1:25 dilution as compared with those in a 1:200 suspension, but the movement in the dense suspension was abruptly stopped by increasing PCOa to 6 %, while the spermatozoa still move in the dilute one when the gas phase contains up to 8 % CO2.
DISCUSSION
The experiments reported above clearly show that CO2 inhibits both respiration and motility of sea-urchin spermatozoa. The results well support our previous assumption, that O2 uptake of the spermatozoa should be much reduced in the absence, as com pared with the presence, of KOH used to absorb CO2) when the suspending medium is sea water (Mohri & Horiuchi, 1961). If the spermatozoa are allowed to respire in a standard Warburg flask without KOH, much respiratory CO2 accumulates both in the gas phase and within the spermatozoa. Although the CO2 tensions showing a marked inhibitory effect are rather high, it is conceivable that respiratory CO2 produced intracellularly is more effective than CO2 added extracellularly. In fact, the results of an experiment tracing the utilization of endogenous phospholipids, which is considered an index of O2 uptake in sea-urchin spermatozoa, showed that the rate of phospholipid utilization slows down greatly after about 30 min. incubation at 20°C. when the respiratory CO2 is allowed to accumulate (Mohri, unpublished). The available data indicate that the inhibitory effect is exerted by gaseous or free CO2, but not by chemically bound CO2. On the other hand, Laser & Rothschild (1939) reported that the respiration rate of sea-urchin eggs when the CO2 is absorbed by KOH is considerably smaller than that without the absorption. They ascribed this reduction in O2 uptake to the comparative absence of bicarbonate ions or to the increase in pH accompanying the absorption of CO2. The situation is, thus, entirely different for eggs and for spermatozoa. This difference should be due to the difference in metabolic pattern.
It is reasonable to assume that CO2 is also a factor involved in the Dilution Effect of sea-urchin spermatozoa, because the inhibitory effect of CO2 is removed by introducing O2 or air. The facts that the suppressing action of CO2, especially on the motility, can be observed more easily in air than in pure O2, and that the effect is more marked in dense sperm suspensions than in dilute ones, are also favourable to this view, although the final conclusion must await the direct measurements of CO2 tension in semen and within the cells. It appears, therefore, that CO2 tension and O2 tension (cf. Rothschild, 1948 a, b), and also pH especially in some sea-urchin species such as E. esculentus (cf. Rothschild, 1956 a, b), are the main factors responsible for the Dilution Effect, i.e. the activation of spermatozoa after dilution, much as Cohn (1918) considered some forty-five years ago. It is unlikely, on the other hand, that CO2 is also responsible for the Respiratory Dilution Effect, i.e. the increase in O2 uptake per unit number of spermatozoa following to dilution of sperm suspensions with constant removal of respiratory CO2. At least at present, trace metals, Cu and Zn, in sea water are the most probable factors causing this effect as described in the Introduction, although further investigation is necessary before this hypothesis can be accepted. There remains, however, the possibility that CO2 exerts its inhibitory effect at least temporarily within the sperm cells, since in the manometric experiments it is not possible to obtain a true CO2 vacuum, but only a steady state in spite of the constant removal by KOH of evolved CO2. Whether CO2 plays some role in senescence or not is obscure at present. Anyway, as correctly pointed out by Rothschild (1948b, p. 366), ‘there is no single factor responsible for senescence, nor for the Dilution Effect’.
The question then arose concerning how and where CO2 exerts its inhibitory effect on sperm activity. In earlier reports, the effect has been called CO2 narcosis. This is merely a description and not an explanation. Recently, Salisbury and his co-workers (Salisbury, VanDemark, Lodge & Cragle, 1960) have shown that a above about 2 % of an atmosphere inhibits glycolysis of bull spermatozoa. The inhibition is also reversible by replacement of CO2 by air or by N2. They assumed that the effect of CO2 in depressing glycolysis rests on its influence on intracellular pH, resulting from rapid penetration of CO2 across the cell membrane. This is also a possible explanation of the inhibitory effect of CO2 on the respiration of sea-urchin spermatozoa. As regards the site of the inhibitory action of CO2 in bull spermatozoa, their results suggest that the uptake of hexose is prevented in a medium containing Na as the predominant cation. This, however, does not occur when the medium contains K in place of Na. In this case the inhibition seems to occur before the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate. Since sea-urchin spermatozoa in contrast to mammalian spermatozoa have at best only a feeble glycolytic machinery (Rothschild, 1948 a; Mohri, 1957), and since the respiration of sea-urchin spermatozoa proceeds through the oxidation of endogenous phospholipids (Rothschild & Cleland, 1952 ; Mohri, 1957), the process of degradation and oxidation of phospholipids would involve the site of the inhibition by CO2. Salisbury (1959) has further shown that sulphide and sulphite are capable of reversing the inhibition of anaerobic glycolysis by CO2, speculating that sulphide and sulphite are produced from sulphhydryl compounds by the action of enzymes such as cysteine desulphhydrase and that they participate in the reaction releasing CO2 from the sperm cells. It is worth while examining whether these compounds are also involved in the mechanism of the Dilution Effect of sea-urchin spermatozoa. Preliminary experiments with these compounds, however, have brought about no definite results. Some other mechanism, therefore, must be involved in the Dilution Effect. In this connexion, the work by Bendall, Ranson & Walker (1960), which showed the inhibition of succinic dehydrogenase by CO2 in a particulate system of Ricinus endosperm, is very suggestive. Experiments along this line are now in progress.
Apart from the problems with spermatozoa, the possible roles of CO2 in many biological phenomena such as cell differentiation and amoeboid movement have recently been emphasized by Loomis (1961). According to him, CO2 tension is the first self-produced regulator in the process of cell differentiation. In this case, too, gaseous or free CO2 is considered as the responsible factor, owing to its ability to pass easily through the cell membrane, and to its high reactivity. It is necessary, therefore, to pay much attention to this naturally occurring metabolic regulator.
ACKNOWLEDGEMENTS
The authors are much indebted to Prof. J. Ishida and to Lord Rothschild for their interest and valuable suggestions. We also thank the Director and Staff of the Misaki Marine Biological Station for supplying sea-urchin materials used in this study. One of us (H. M.) is grateful to Dr P. Dohrn for allowing him to use the facilities of the Stazione Zoológica, Naples. The technical assistance of Miss T. Abe and Mrs T. Mohri is also acknowledged. Finally, we are indebted to Dr A. Packard for his kind help in preparing the manuscript.