The Respiration of Sea-Urchin Spermatozoa

1950 ◽  
Vol 27 (3) ◽  
pp. 420-436
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Oxygen Saturation ◽  
Sea Urchin ◽  
Seminal Plasma ◽  
Sea Water ◽  
Chelating Agent ◽  
Dilution Effect ◽  
O2 Uptake ◽  
Sperm Density ◽  
Dense Suspensions ◽  
Mammalian Spermatozoa

1. The O2 uptake of sea-urchin spermatozoa, Echinus esculentus, has been reexamined under varying conditions of sperm density, and in the presence of CuCl22H2O and sodium diethyldithiocarbamate (DDC). 2. The total O2 uptake of dilute sperm suspensions was previously thought to be higher than that of dense suspensions per unit quantity of spermatozoa (Dilution Effect I). This result is only obtained when the oxygen saturation of the dense suspension is inadequate, which may easily occur as the QOO2 of sea-urchin spermatozoa may reach 30 at 15.0° C. When oxygen saturation is satisfactory, the total O2 uptake of dense solutions is slightly greater than that of dilute ones. The experiment cannot be done in micro-respirometers of the normal Warburg type unless the density of spermatozoa per ml. suspension is less than about 109 corresponding to an initial semen dilution of 1:20 or 1:25. These figures apply to other manometric experiments on the O2 uptake of sea-urchin spermatozoa using normal amounts of material. 3. When movement ceases, there is a sharp increase in the O2 uptake of the suspension. 4. The addition of seminal plasma to dilute sperm suspensions does not inhibit the increased rate of O2 uptake, per unit quantity of spermatozoa, observed in these suspensions when compared with dense ones (Dilution Effect II). Dilution Effect II is therefore not caused by the dilution of an inhibitory substance in seminal plasma. 5. Sperm suspensions were prepared by diluting semen 1:50 with sea water and allowing them to respire for 45 mm. They were then centrifuged, the supernatant was discarded and the spermatozoa were re-suspended to different densities with sea water. This treatment has the following effects: (a) Centrifugation irreversibly damages the spermatozoa and reduces their O2 uptake. (b) Removal of the supernatant, which contains seminal plasma, and re-suspension in sea water also reduces O2 uptake. (c) The treatment markedly reduces Dilution Effect II. If the experiment is done in the same way but the suspensions are only allowed to respire for 10 min. before centrifugation, (a) and (b) are the same, but Dilution Effect II is normal. This shows that during metabolism, a regulatory substance is lost from dilute suspensions, as in mammalian spermatozoa; but this is not the cause of Dilution Effect II. 6. Dilution Effect II, considered as the reduced O2 uptake of dense suspensions, can be reversed by the addition of CuCl22H2O, 1 p.p.m., to the medium. 7. Dilution Effect II can be made to occur in sperm suspensions which do not normally exhibit it, by the addition of DDC, in concentrations as low as 3.64x10-5M (final concentration). The action of DDC is not greater when its concentration is increased to 10-3M, which suggests that in these conditions it acts as a chelating agent and not as a narcotic. For the same reasons its oxidation product, tetra ethyldithiocarbamyl disulphide, is unlikely to be responsible for DDC's inhibitory effect on sperm O2 uptake. 8. These results are consistent with the hypothesis that Dilution Effect II is due to the amounts of copper (or possibly zinc) in sea water being inadequate to satisfy the requirements of dense sea-urchin sperm suspensions. This situation is unlikely to arise during natural spawning as sperm densities are too low for the effect to occur in these conditions. Other interpretations of the stimulating action of copper and zinc are discussed. 9. The experiments remove several of the differences hitherto believed to exist between sea-urchin and mammalian spermatozoa.

The Physiology of Sea-Urchin Spermatozoa

1950 ◽  
Vol 27 (1) ◽  
pp. 59-72
Author(s):  
LORD ROTHSCHILD ◽  
P. H. TUFT
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Dilution Effect ◽  
O2 Uptake ◽  
Time Curve ◽  
Sperm Density ◽  
Negative Results ◽  
Catalytic Function ◽  
Sperm Suspension ◽  
Dense Suspensions

1. When a suspension of sea-urchin semen in sea water is diluted, the total O2 consumed and the rate of O2 uptake per spermatozoon are greater than before dilution. This is the Dilution Effect, first described by Gray (1928 a). A further investigation of this phenomenon has been made, using the semen of Echinus esculentus. 2. The form of the O2 uptake-time curve of such suspensions varies according to the ratio semen: sea water. 3. In very dense suspensions (d ≥ 5 x 109 sperm/ml.), the apparent low O2 uptake per unit quantity of spermatozoa is mainly due to inadequate O2 saturation of the lower layers of the suspension, in which the spermatozoa are virtually unable to respire. Such suspensions are not suitable for experiments in Warburg or Barcroft manometers. 4. In dense suspensions (4 x 108 < d ≤ 109 sperm/ml.) the Dilution Effect was observed when sea water was added to the suspension, in such proportions that the sperm density was only reduced by a factor of 1.14. No Dilution Effect occurred when isotonic ‘Analar’ NaCl was added in the same proportions. On the basis of the small dilution involved, and the negative results with ‘Analar’ NaCl, it is concluded that the Dilution Effect is not exclusively due to the sperm having more space to move after dilution and therefore being able to expend more energy. 5. The Dilution Effect occurs when isotonic ‘Analar’ NaCl containing 1 p.p.m. CuCl2 is added to a dense sperm suspension, in the above proportions. Stimulation of O2 uptake is proportional, over certain limits, to the amount of CuCl2 added. Both CuCl, ZnCl2, and to a lesser extent CuSO2, have similar effects. 6. In a particular experiment, egg-water and Cu salts were about equally efficacious in increasing the O2 uptake of a sperm suspension. 7. The characteristic decline in the O2 uptake of dense suspensions can be partly reversed by the addition of Cu salts. 8. In dilute suspensions (d < 4 x 108 sperm/ml.), there is no Dilution Effect when sea water or CuCl2 are added in the above proportions. On the other hand, the decline in O2 uptake can be partly arrested by the addition of Cu salts. 9. Diethyldithiocarbamate markedly inhibits the O2 uptake of unwashed sea-urchin spermatozoa diluted with sea water. The inhibition does not take place in the presence of added CuCl2. Cu may therefore have a catalytic function in the metabolism of sea-urchin semen. 10. The possibility that Cu lack is responsible for the reduced O2 uptake of spermatozoa in dense suspensions is considered.

scholarly journals The Physiology of Sea-Urchin Spermatozoa

1956 ◽  
Vol 33 (1) ◽  
pp. 155-173
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Usnic Acid ◽  
Dilution Effect ◽  
O2 Uptake ◽  
Sperm Suspension ◽  
Dense Suspensions ◽  
Cast Doubt ◽  
Dilute Suspensions ◽  
Glycyl Glycine

1. Sea-urchin spermatozoa (Echinus esculentus) are extremely sensitive to changes in the pH of the suspending medium, their respiration being proportional to pH between 7.6 and 8.4. 2. In a manometric experiment in which semen was diluted 1/25 with sea water (CO2 absorbed), the pH of the suspension was 7.5 at the beginning of the experiment and 8.2 at the end, after 180 min. incubation at 15° C. 3. Sea water, buffered with glycyl glycine, 0.025 M and brought to pH 8.3, was added to a sperm suspension whose pH was 7.5 at the beginning of the experiment, after a period of incubation. There was a pronounced respiratory Dilution Effect. When the sea water was buffered with glycyl glycine and brought to pH 7.8, and this diluent was added to the same sperm suspension, there was a negligible respiratory Dilution Effect. 4. The O2 uptake of suspensions prepared in buffered sea water at pH 8.3 was markedly higher than that in the same buffered sea water at pH 8.0. 5. These observations cast doubt on the reality of the respiratory Dilution Effect when observed in experiments in which sea water is the suspending medium and respiration is measured by a method involving the absorption of CO2. An exception to this generalization was observed in buffered sea water at a low pH, 7.6. In this case, the O2 uptake of dilute suspensions was greater, per unit number of spermatozoa, than that of dense suspensions. 6. 2,4-dinitrophenol, 6 x 10-5M, stimulated the O2 uptake and depressed the motility of spermatozoa more in dense than in dilute suspensions. Versene, 10-3M, reversed the action of 2,4-dinitrophenol. 7. The results of other workers on adding suspensions of usnic acid to sea-urchin spermatozoa were not confirmed. This substance is more toxic to spermatozoa in dilute than in dense suspensions.

The Physiology of Sea-Urchin Spermatozoa

1948 ◽  
Vol 25 (4) ◽  
pp. 353-368
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Protective Action ◽  
Wave Length ◽  
Dilution Effect ◽  
O2 Consumption ◽  
O2 Uptake ◽  
Sperm Suspension ◽  
Dense Suspensions ◽  
Dilute Suspensions

1. An investigation has been made into the causes of the interrelated phenomena of Senescence and the Dilution Effect in the spermatozoa of Echinus esculentus. The Dilution Effect is the increase in O2 uptake and in movement observed when a concentrated sperm suspension, obtained by partial dilution of semen, is diluted with sea water. 2. The supernatant sea water obtained by centrifuging partially metabolized sperm suspensions contains no substances which depress the respiration or motility of the spermatozoa of the same species. This supernatant sea water has a small but definite protective action on spermatozoa in maintaining but not increasing their O2 consumption. The effect is enhanced by agitating the suspensions before centrifugation. 3. E. esculentus spermatozoa do not glycolyse aerobically, anaerobically, or in the presence of 5% O2 in CO. The absence of aerobic acid production proves that the reduced O2 uptake per sperm of dense suspensions is not due to a lowering of the pH of the medium. 4. As the Dilution Effect is observed in manometric experiments in which CO2 is continuously and adequately removed, neither accumulation of CO2 nor a reduction in pH resulting from the accumulation of CO2 are responsible for the reduced O2 uptake of dense suspensions. 5. The O2 uptake of dense sperm suspensions (> 109 sperm/ml.) is altered by changes in O2 tension. O2 consumption in 100% oxygen is nearly double that in 10% O2 in N2. An increase in O2 tension has no stimulating action on dilute suspensions. 6. An atmosphere of 100% O2 has a gradual toxic effect on dense and dilute sperm suspensions. 7. Carbon monoxide has a greater inhibitory action on dilute than on concentrated sperm suspensions. 8. The difference between the effects of varying O2 tensions or CO on dense and dilute suspensions is partly, or perhaps wholly, due to gaseous diffusion being a limiting factor. This may partly explain the Dilution Effect. 9. Apart from photo-reversibly inhibiting respiration, CO exerts an irreversible toxic influence on sea-urchin spermatozoa. 10. Illumination of spermatozoa with a wave-length of light outside the region of photo-chemical absorption by CO cytochrome oxidase enables visual examination to be made in the presence of CO. The O2 uptake of a suitable concentration of spermatozoa is 70% inhibited by 5% O2 in CO. The same inhibition occurs when the suspension is illuminated by light of wave-length 548 mµ, but there is no equivalent decrease in motility. 11. After motility and respiration have ceased sea-urchin spermatozoa still contain oxidizable substrates and the appropriate dehydrogenases as evidenced by their ability to reduce methylene blue in an evacuated Thunberg tube.

The Physiology of Sea-Urchin Spermatozoa Action of Versene

1954 ◽  
Vol 31 (2) ◽  
pp. 252-259
Author(s):  
LORD ROTHSCHILD ◽  
A. TYLER
Keyword(s):  
Trace Metals ◽  
Sea Urchin ◽  
Seminal Plasma ◽  
Sea Water ◽  
Dilution Effect ◽  
Dense Suspension

1. The effect of adding sea water containing different concentrations of versene to suspensions of sea-urchin spermatozoa (Echinus esculentus) has been investigated, as regards their respiration, motility and fertilizing capacity. 2. The respiratory Dilution Effect is progressively reduced and finally abolished when, instead of sea water, sea water containing 10-6, 10-5, 10-4 or 10-3 M versene is added to sperm suspensions. 3. At the same time versene greatly delays the senescence of the spermatozoa, both as regards their motility and fertilizing capacity. For example, after seventeen hours, a 105 sperm/ml. suspension in sea water containing 10-3 M versene has 125 times the fertilizing capacity of a suspension containing 107 sperm/ml. without versene. 4. The change in the ratio of seminal plasma to sea water which occurs when a dense suspension is diluted does not explain the Dilution Effect. 5. These results are discussed in relation to the hypothesis which accounts for the Dilution Effect in terms of trace metals, particularly copper, normally occurring in sea water, and the amounts available per spermatozoon under various conditions of dilution.

The Physiology of Sea-Urchin Spermatozoa

1950 ◽  
Vol 26 (4) ◽  
pp. 396-409
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Seminal Plasma ◽  
Human Blood ◽  
Phosphate Buffer ◽  
Dry Weight ◽  
O2 Uptake ◽  
Low Concentrations ◽  
Initial Substrate ◽  
Rough Calculation ◽  
Initial Substrate Concentration

1. Spermatozoa and seminal plasma of Echinus esculentus contain catalase. 2. At 15° C., 4 ml. of a suspension of semen diluted with neutral phosphate buffer in the ratio 1:13 produced in 1 min. 90µl. O2 from an H2O2 solution containing 150 µl. O2. The dry weight of semen in the suspension was 45 mg. and the number of spermatozoa 8.55x109. Under the same conditions, seminal plasma obtained by centrifuging semen produced 50 µl. O2 in 1 min. The dry weight of seminal plasma in the suspension was 12 mg. Human blood, dry weight 229.3 mg./ml., must be diluted with phosphate buffer in the ratio 1:1700 to produce the same amount of O2 in 1 min. as the above suspension of semen. If catalatic activity is defined by the equation Ac = (gt)-1 In {a/(a-x)}, where g = weight in g./ml. of the catalase-containing material, t = 1 min., a = initial substrate concentration (H2O2), and x = amount of H2O2 decomposed in 1 min. at 15° C., Ac = 80-100, 150-200 and 6800 respectively for sea-urchin semen, sea-urchin seminal plasma and human blood. 3. The catalatic activity of semen and seminal plasma is strongly inhibited by hydroxylamine. 4. The O2 uptake and motility of sea-urchin spermatozoa is unaffected by M/5000 H2O2. Higher concentrations of H2O2, M/3000-5000, produce a pronounced ‘shock’ effect, from which the spermatozoa often completely recover. 5. Low concentrations of hydroxylamine, M/3000, reduce O2 uptake and motility. 6. Sea-urchin spermatozoa are almost instantly killed by combinations of hydroxylamine and H2O2, at concentrations which are relatively innocuous when the substances are added separately. 7. A rough calculation indicates that a single spermatozoon contains less than 500 molecules of catalase. 8. A new method of adding H2O2 to catalase-containing material in a manometer is described.

The Physiology of Sea-Urchin Spermatozoa

1948 ◽  
Vol 25 (4) ◽  
pp. 344-352
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Seminal Plasma ◽  
Sea Water ◽  
O2 Activation ◽  
Residual Tension ◽  
K Concentration

1. Echinus esculentus spermatozoa are normally motionless in undiluted semen. They become active when the semen is diluted with sea water or seminal plasma obtained by gentle centrifugation (1500 r.p.m., 12 cm. radius, for 15 min.). 2. A sperm-immobilizing substance can, however, be obtained in seminal plasma by more prolonged centrifugation of semen. 3. Spermatozoa can be made motile in undiluted semen by increasing the O2 tension in the atmosphere surrounding the semen. 4. This O2 activation is completely inhibited by N2; the N2 effect is reversible. 5. Measurements of seminal O2 tension were made with an O2 electrode. The O2 tension of semen is low, being at most 15 mm. Hg. It was not possible to decide whether this residual tension was due to O2 or some other substance reacting at the electrode. 6. The K content of seminal plasma is about 1·55 mg./ml., which is four times higher than that of perivisceral fluid or sea water. 7. The pH of semen is lower than that of sea water, being approx. 7·5. 8. Neither K concentration nor pH is responsible for the inactivity of sperm in semen. 9. A hormone, Androgamone I, is often considered to be responsible for the inactivity of spermatozoa in non-mammalian semen. No support has been found for this view and it is concluded that in E. esculentus semen the spermatozoa are motionless through lack of O2.

Studies on the Respiration of Sea-Urchin Spermatozoa: III. Respiratory Quotient

1961 ◽  
Vol 38 (2) ◽  
pp. 249-257
Author(s):  
H. MOHRI ◽  
K. HORIUCHI
Keyword(s):  
Sea Urchin ◽  
Respiratory Quotient ◽  
Sea Water ◽  
Direct Method ◽  
O2 Uptake ◽  
Present Material ◽  
A Value ◽  
Sperm Suspension ◽  
Co2 Output ◽  
The Difference

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 C02. 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.

The Physiology of Echinus Esculentus Spermatozoa

1948 ◽  
Vol 25 (1) ◽  
pp. 15-21
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
O2 Uptake ◽  
Reversible Effect ◽  
Spectroscopic Examination

1. The O2 uptake of Echinus esculentus sperm suspensions is strongly inhibited by CO. In equilibrium with a mixture of 90% CO and 10% O2, the O2 uptake is 60% less than that of an identical suspension in equilibrium with 90% N2 and 10% O2. 2. This inhibition is completely reversed by light. 3. Spectroscopic examination revealed cytochromes a, a3, b and c. On treatment with CO the spectrum of COa3 was observed. 4. These results establish that the cytochrome system is the catalytic agent which controls the respiration of sea-urchin spermatozoa. 5. The O2 uptake of sperm suspensions in equilibrium with 90% N2 and 10% O2 is about 10% lower than in air. 6. Certain difficulties in measuring the R.Q. of sperm are discussed. 7. The reversible effect of CO on the O2 uptake of spermatozoa is the same whether they are in egg water or sea water.

A Note on the Copper Content of Sea-Urchin Semen and Sea Water

1950 ◽  
Vol 27 (2) ◽  
pp. 123-125
Author(s):  
H. BARNES ◽  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Seminal Plasma ◽  
Copper Content ◽  
Water Surface ◽  
Sea Water ◽  
Sea Urchins ◽  
Coelomic Fluid ◽  
Original Sample

1. The copper content of semen, seminal plasma and coelomic fluid of Echinus esculentus has been determined. The average figures for sea-urchins examined immediately after collection were: semen, 1.33µg/ml.; seminal plasma, 0.50µg/ml.; coelomic fluid, 0.07µg./ml. The accuracy of the method of estimation was ± 0.08µg. Cu/ml. original sample, which means that the amount of Cu in coelomic fluid is not significant. This figure for coelomic fluid is considerably lower than that obtained by Webb (1937) in the only previous examination. 2. The copper content of sea water (surface, off Keppel Pier, Millport) was 0.0064µg./ml.

The Fertilization Reaction in the Sea-Urchin

1953 ◽  
Vol 30 (1) ◽  
pp. 57-67
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Sea Water ◽  
Interaction Time ◽  
Conduction Time ◽  
Incomplete Block ◽  
O2 Uptake ◽  
Sharp Reduction ◽  
Sea Urchin Eggs

1. Treatment of unfertilized sea-urchin eggs with sea water containing nicotine is known to induce polyspermy when the eggs are subsequently inseminated with homologous spermatozoa, at densities which cause a very small amount of polyspermy in untreated eggs. 2. If nicotine were to increase the speeds at which sea-urchin spermatozoa swim, the chances of fertilization, and therefore of polyspermy, might be increased. Nicotine does not increase sperm speeds; in addition, it causes a sharp reduction in the O2 uptake of these spermatozoa. 3. The only other ways in which nicotine could induce polyspermy are by altering the egg surface in such a way that the probability of fertilization is increased by a factor of about twenty; or by lengthening the conduction time of the block to polyspermy. Experiments described in this paper show that the first explanation is untenable and therefore that the second is the correct one. It is concluded that nicotine abolishes the fast incomplete block to polyspermy and that over-exposure to this substance probably abolishes the block to polyspermy altogether. 4. Polyspermic eggs divide sooner than monospermic ones. 5. When, as in these experiments, eggs are allowed to interact with spermatozoa for known times, and then functionally separated by immersion in hypotonic sea water, some eggs, presumably those which sustain a successful sperm-egg collision at the end of the interaction time, are activated but not fertilized by the spermatozoon, as in the pseudogamous nematodes. Cleavage does not occur though the egg nucleus swells. 6. Previous results in the same field and observations by other workers are discussed.

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