Some correlations between development and respiration in the sand dollar egg,as shown by cyanide inhibition studies

Cyanide is a valuable tool for studying respiratory mechanisms and their r?le in embryonic development: it is relatively specific in its action, penetrates cell membranes readily, is active in low concentration, and may be controlled quantitatively (page 217). Echinarachnius is extremely sensitive to cyanide and the oxygen consumption of both eggs and of sperm is almost completely inhibited by 10(-5)M HCN (pages 219 and 221). Cell division is likewise arrested by the same concentration (page 223). One of the pronounced effects of an irreversible dosage of cyanide is the marked cytolysis or breakdown of the egg, both internally and at the cell membrane. This cytolysis appears to be related to the state of metabolism, and its occurrence varies with both the respiratory and developmental activity of the cell (page 224). The lethal dosage of cyanide varies with the state of development of the egg: the unfertilized egg is less susceptible than the fertilized one, and the susceptibility increases as the development of the fertilized egg proceeds (page 228). The Echinarachnius egg differs from that of Arbacia in respiratory behavior chiefly in its inability to survive prolonged anoxia: the sea urchin egg will tolerate for 24 hours a concentration of cyanide that kills the sand dollar eggs in 30 minutes (page 229). The Echinarachnius egg is apparently completely dependent upon cyanide-sensitive catalytic systems for its normal functioning and maintenance. Interference with this aerobic energy release mechanism results in irreversible damage to the egg (page 231).     

Respiratory Mechanisms in the Aroid Spadix

The flowers of Skunk-cabbage (Symplocarpus foetidus), like thespadix tissues of other Aroids, have a rapid, carbon monoxideand cyanide (HCN) resistant respiration; oxygen uptake is independentof the oxygen partial pressure over a wide range. Cell fractionswere isolated by differential centrifugation and their oxidativeactivities studied. Oxidation of succinate and citrate by mitochondriacan be inhibited 50 to 60 per cent. by 1 X 10^–3 M. HCN,and antimycin A (AA) causes partial inhibitions. An active mitochondrialcytochrome-c oxidase is present, and it shows a typical sensitivityto cyanide. The mitochondria possess an active reduced diphosphopyridine-nucleotide(DPNH) oxidase system, which is inhibited roughly 80 per cent.by 1 X 10^–3 M. HCN and 1.7 µg./ml. AA. The microsomalDPNH oxidase, which is less sensitive to inhibitors, is lessactive per gramme of tissue than that on the mitochondria. Thefinal supernatant shows little DPNH oxidase. With all fractions,reduced triphosphopyridine nucleotide (TPNH) is oxidized muchmore slowly than DPNH. DPNH-cyto-chrome-c reductase activitywas measured; the mitochondrial system is partially blockedby AA, whereas the microsomal activity is AA-insensitive. Spectro-photometricexamination of a preparation of solubilized mitochondria showedthat cytochromes a, b, and c are present. The results are discussedwith reference to the pathway and localization of hydrogen andelectron transport in the Aroid spadix.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : V. CYTOCHROME OXIDASE ACTIVITY IN THE EGGS OF ARBACIA PUNCTULATA

1. An enzyme capable of oxidizing reduced cytochrome c (i.e. a cytochrome oxidase) has been obtained from Arbacia eggs. In 0.02 M hydroquinone, the cytochrome oxidase was half activated at a cytochrome c concentration of approximately 4 x 10^–6 M. The concentration of the cytochrome oxidase was found to be nearly the same in unfertilized and fertilized eggs, the amount of the enzyme—as measured by means of its activity toward cytochrome c as a representative substrate—being more than sufficient to account for the highest rate of oxygen utilization yet observed in the intact, living, fertilized eggs, and of the same order as that in certain rat tissues. 2. The Arbacia cytochrome oxidase was strongly inhibited by carbon monoxide in the dark, the inhibition being almost completely reversed by light. The inhibition constant was not greatly altered by variation in the concentration of cytochrome c or the concentration of hydroquinone used as reductant for the cytochrome c, having a value of 3 to 5 under the conditions used. The inhibition constant was about 2 with p-phenylenediamine as reductant for the cytochrome c, but apparently had the surprisingly low value of about 0.5 with 0.02 M cysteine as reductant. 3. The cytochrome oxidase was completely inhibited by sufficiently high concentrations of sodium cyanide, sodium azide, and sodium sulfide. It was also completely inhibited in 0.6 M sodium chloride. It was not inhibited by two inhibitors of copper containing enzymes, 8-hydroxyquinoline and sodium diethyldithiocarbamate. It was also not significantly inhibited by 2,4-dinitrothymol, 2,4-dinitro-o-cyclohexylphenol, phenylurethane, 5-isoamyl-5-ethylbarbituric acid, or iodoacetic acid. 4. Quantitative examination of the fertilized eggs showed that cytochrome c, if present at all, occurred in a concentration of less than 2 micrograms per gram of wet fertilized Arbacia eggs. On the basis of these data and those of Fig. 2, above, it seems safe to conclude that cytochrome c cannot carry a significant fraction of the oxygen consumption of fertilized Arbacia eggs. It was also found that, in contrast to similar preparations from certain other animal tissues, the Arbacia cytochrome oxidase preparation displayed no succinic dehydrogenase activity when tested manometrically in the presence of excess cytochrome c. 5. Extending previously reported (3) experiments with other inhibitors, the effects of sodium azide and sodium sulfide on the respiration and cell division of fertilized Arbacia eggs were determined, the eggs being initially exposed to the reagents 30 minutes after fertilization at 20°C. With either reagent cleavage was completely blocked by a concentration of reagent which reduced the respiration to approximately 50 per cent of the normal level. 6. On the basis of certain theoretical considerations regarding the possible mechanism of action of cyanide and other respiratory inhibitors it is suggested that a fraction of the respiration apparently concerned with supplying energy for division processes in the fertilized Arbacia egg may be keyed into the respiratory cycle through a carrier having a somewhat higher potential than those which carry the larger portion of the egg respiration. The theory is also employed in an effort to resolve a number of hitherto apparently paradoxical observations regarding the effects of cyanide, azide, and carbon monoxide on cell respiration.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiration of spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , was found to be sensitive to rotenone, antimycin A, and cyanide. This suggests that sperm respiration results from electron transport which spans the whole mitochondrial respiratory chain. The sperm respiration was inhibited by oligomycin and this inhibition was released by 2, 4-dinitrophenol (DNP). DNP did not stimulate the respiration of spermatozoa in a diluted suspension (2 × 10⁸/ml), where they were swimming vigorously. The ADP level of spermatozoa in the diluted suspension was markedly higher than that in dry sperm. The spermatozoa, which had reacted with unfertilized eggs fixed with glutaraldehyde, were immotile with a quite low respiratory rate. The respiratory rate of the immotile spermatozoa was enhanced by DNP. In the immotile spermatozoa, ADP level was markedly low and the ATP level was as high as that in dry sperm. From these findings, it is concluded that in the swimming spermatozoa respiration coupled with oxidative phosphorylation occurs at the maximum rate. State 3 respiration probably occurs in the swimming spermatozoa. The low respiratory rate of the immotile spermatozoa is assumed to be due to a shortage of ADP and is practically regarded as state 4 respiration.     

Silicon metabolism in diatoms. III. Respiration and silicon uptake in Navicula pelliculosa

1. Evidence is presented that silicon uptake in the diatom Navicula pelliculosa is linked with aerobic respiration. 2. Cyanide, fluoride, iodoacetate, arsenite, azide, and fluoroacetate, at concentrations inhibitory to respiration, were also inhibitory to silicon uptake. 3. 2,4-Dinitrophenol (1 to 2 x 10(-5)M) stimulated respiration by 100 per cent, but almost completely inhibited silicon uptake. 4. The respiratory quotient of non-Si-deficient cells decreased from 0.93 to 0.75 after 4 days of starvation in darkness. Glucose (1 per cent) raised the respiratory quotient of such starved cells to 1.05. 5. Silicate (20 mg. Si/liter) stimulated respiration of unstarved Si-deficient cells by about 40 per cent. The effect of silicate on the respiration of Si-deficient cells which had been starved in darkness for 4 days was less marked. 6. The respiratory quotient of Si-deficient cells decreased from 0.8-0.9 to 0.3 after 4 days of starvation in darkness. The addition of silicate to starved cells raised the quotient to 0.5. This represented a 25 per cent stimulation of oxygen uptake concomitant with a 90 per cent stimulation of carbon dioxide evolution. 7. Glucose (1 per cent) caused an increase of respiratory quotient in starved cells from 0.3 to 0.7-0.8. The addition of silicate had no effect on the R.Q. during the oxidation of exogenous glucose. 8. Substrates (glucose, fructose, galactose, lactate, succinate, citrate, glycerol), which caused a stimulation of respiration in starved cells, also stimulated silicon uptake by those cells. However, the stimulation of silicon uptake (50 to 100 per cent) was not proportional to the respiratory stimulation by these substrates (30 to 300 per cent).     

Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

Speract. Purification and characterization of a peptide associated with eggs that activates spermatozoa

A low molecular weight peptide (speract) associated with sea urchin eggs has been purified to apparent homogeneity by charcoal adsorption, DEAE-Sephacel chromatography, Bio-Gel P-2 filtration, and Dowex AG 50W-X4 chromatography. Gametes from 5000 female sea urchins were required for the isolation of approximately 9 mg of the peptide. The isolated peptide is homogenous based on [3H]acetic anhydride labeling, gel filtration, and reverse phase high pressure liquid chromatography. Speract is composed entirely of neutral and acidic amino acids with glycine as the major component, and it appears to have a blocked NH2 terminus based on its insensitivity to leucine aminopeptidase, its failure to react with dansyl chloride, and its chromatographic behavior on strong cation exchange resins. Speract is a potent stimulator of sea urchin sperm oxygen consumption, causing significant increases of sperm respiration rates at concentrations as low as 10(-12) M and producing 20-fold increases of oxygen consumption at maximal concentrations of 10(-8) M. Sperm cyclic GMP and cyclic AMP concentrations are also increased by speract, but concentrations of at least 10(-10) M and 10(-9) M are required for half-maximal elevations, respectively. The peptide, purified from Strongylocentrotus purpuratus eggs, also cross-reacts with spermatozoa from Lytechnis pictus sea urchins, suggesting that speract does not show species specificity. These results represent the first report of the purification of a peptide associated with eggs that may affect spermatozoa under natural conditions.     

Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.     

Change in the Respiratory Rate of Sea Urchin Spermatozoa following Sperm-egg Interaction in the Absence of Mg2+

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus (10⁸ cells/ml), preincubated with unfertilized eggs deprived of jelly coats (more than l0⁵ cells/ml) at 20°C for 20min in Mg²⁺ free artificial sea water containing 1 mM Ca²⁺ (MFASW), exhibited very low respiration, which was enhanced by 2, 4 dinitrophenol (DNP). The fertilization rate in MFASW was usually less than 5% and was about 25% at most. Preincubation with fertilized eggs (with and without a fertilization membrane) in MFASW did not reduced the respiratory rate of spermatozoa. The rate of sperm respiration was lower in MFASW than in artificial sea water (ASW), but was higher than the respiratory rate of spermatozoa preincubated in MFASW with unfertilized eggs. Sperm respiration in MFASW or in ASW was not stimulated by 2, 4 dinitrophenol. Almost complete inhibition of sperm respiration was obtained with unfertilized eggs fixed with glutaraldehyde at concentrations of above 10⁵ cells/ml in MFASW and of about l0⁴ cells/ml in ASW. The respiratory rate of spermatozoa treated with fixed eggs was enhanced by DNP. It is concluded that the respiratory rate of the spermatozoa is reduced by their interaction with unfertilized eggs before their penetration into the eggs.     

The change in osmotically inactive fraction produced by cell activation

1. Resting and activated eggs of the sea urchin Arbacia punctulata were swollen in hypotonic sea water (60, 70, 80, and 90 per cent), and allowed to attain equilibrium volumes (Figs. 1 and 2). 2. Both fertilized and unfertilized eggs obey the Boyle-van't-Hoff law, but the value for b, the "osmotically inactive fraction" or non-swellable volume, was different for the two, averaging in the cases studied 7.3 per cent for unfertilized and 27.4 per cent for fertilized. 3. On activation, the eggs of the sea urchin undergo a definite increase in total cell volume, of approximately 2.7 per cent. 4. Some evidence is adduced for the possibility that the alteration in cell volume and in o.i.f. may depend upon the species in question. 5. A parallelism between change in b and alteration of respiratory metabolism in Arbacia, Chaetopterus, and Arbacia fragments is pointed out. This requires further investigation in other species to establish generality. 6. Equations for the calculation of the point at which osmotic pressures and cell volumes are identical for unfertilized and fertilized eggs are included. 7. A mechanical analogue of the phenomena is introduced (Fig. 3).     

THE EFFECTS OF URETHANE AND CHLORAL HYDRATE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

The effects of a series of concentrations of the narcotics, ethyl carbamate and chloral hydrate, have been determined on the consumption of oxygen by fertilized and unfertilized eggs of the sea urchin Arbacia punctulata. In the fertilized eggs the effects of the two inhibitors on cell division were also examined. The following observations were made: 1. Assuming that the narcotic acts upon a single catalyst in the unfertilized egg the degree to which the consumption of oxygen is inhibited in this resting cell can be related to the narcotic concentration by an expression derived from the law of mass action. 2. To account for the relation between the concentration of the narcotic and its effect on respiration in the fertilized eggs, it is necessary to conclude that in them the narcotic acts on two parallel respiratory systems. The experimental data can be quantitatively predicted (1) if the reaction of the narcotic on the two systems is governed by the law of mass action and (2) if 40 per cent of the oxygen consumption is mediated by one system, the "activity" system, and the remainder by the other, the "resting" or "basal" system. 3. The mass law constants applying to the resting system in the fertilized egg are similar to those for the single system functioning in the unfertilized egg so that these two respiratory systems are probably identical. 4. The concentrations of the narcotics just sufficient to abolish cell division affect primarily the activity system, the existence of which was inferred from the respiratory experiments. It is concluded that normal cell division requires specifically the normal function of the activity system, that in fact the energy for cell division is made available through that system.     

Sea urchin sperm peroxidase is competitively inhibited by benzohydroxamic acid and phenylhydrazine

Sea urchin sperm contain a phenylhydrazine-sensitive peroxidase that is believed to use hydrogen peroxide produced by the fertilized egg to reduce sperm fertility and thereby assist in the prevention of polyspermy. Strongylocentrotus purpuratus sperm were treated initially with hypotonic phosphate buffer (pH 7.0) to remove catalase and then extracted with 0.5% Triton X-100 in 0.5 M acetate buffer (pH 5.0). Peroxidase activity in this detergent extract was assayed using 3,3',5,5'-tetramethyl benzidine (TMB) as oxidizable substrate. Kinetic studies showed that the Km for TMB is 250 microM. Benzohydroxamic acid and phenylhydrazine are known to be competitive inhibitors of a variety of plant and animal peroxidases. These substances were found to competitively inhibit the sea urchin sperm peroxidase: for benzohydroxamic acid, Ki = 51.2 microM, mean inhibitory dose (ID50) = 146.7 microM; for phenylhydrazine, Ki = 201 nM, ID50 = 303 nM. These findings indicate that the biochemical properties of the sea urchin sperm peroxidase resembles those of peroxidases found in somatic tissues where oxygen radicals are produced by phagocytes to kill bacteria and support our hypothesis that the sperm peroxidase has a functional role in the prevention of polyspermy during fertilization.     

Respiration of Sea Urchin Spermatozoa in the Presence of a Synthetic Jelly Coat Peptide and Ionophores

Sperm respiration and motility of the sea urchin Hemicentrotus pulcherrimus were studied at pH 6.8 in the presence of a synthetic jelly peptide (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and monovalent cationic ionophores. The synthetic peptide stimulated sperm respiration and motility to the level of that found in normal sea water (pH 8.2) with half-maximal stimulation at approximately 100 pM. Monensin and valinomycin also stimulated sperm respiration with half-maximal effects at 7 μM and 0.7 μM, respectively. The stimulation of sperm respiration by the peptide and monensin was dependent on external Na⁺, but was not dependent on the osmolarity of the suspending medium. Approximately 50 mM Na⁺ was required for half-maximal respiratory responses to the peptide and monensin.     

THE EFFECT OF PENICILLIN ON EGGS OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. Penicillin in the range of concentration from 250 U/ml. to approximately 2650 U/ml. inhibits the rate of cell division of the fertilized sea urchin egg from 0 to 100 per cent. 2. Penicillin in the same range of concentrations has no effect on the oxygen consumption of the unfertilized or the fertilized eggs. 3. Penicillin is bound by some component of the sea urchin egg in amounts sufficiently large to lower the initial concentration, this binding apparently not being related to the inhibitory action.     

Purification and characterization of calmodulin from sea urchin spermatozoa

Calmodulin was purified to apparent homogeneity from sea urchin spermatozoa by heat-treatment at 85 degrees C, ammonium sulphate precipitation at pH 4.2, DEAE-Sephacel chromatography and gel filtration on Sephadex G-100. Approximately 8.3 micrograms calmodulin were recovered per 10(10) sperm cells. The sperm calmodulin had an apparent molecular weight of 17 800. The purified calmodulin activated calmodulin-deficient phosphodiesterase from pig coronary arteries, with half-maximal activation occurring at approximately 40 ng calmodulin/ml. Trifluoperazine also inhibited the sperm calmodulin activity. These results demonstrate that calmodulin is present in high amounts in sea urchin spermatozoa, and that it is essentially the same as the calmodulin isolated from various other tissues.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWIGN INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus , became quite low and spermatozoa was immotile, after sperm suspension containing glutaraldehyde-fixed eggs of homologous species was stirred at 20°C for 15 min. The respiratory rate of fresh spermatozoa, introduced to the suspension of immotile spermatozoa thus obtained, was also reduced markedly. The respiration of fresh spermatozoa was not inhibited by adding them to suspension of intact or acrosome reacted spermatozoa. A heat stable and non-dialyzable substance, which inhibited sperm respiration, was removed from the fixed eggs by vigorously stirring the egg suspension for 10 min, when unfertilized eggs were fixed with insufficient amount of glutaraldehyde (10 ml of 1% glutaraldehyde solution to 1 ml egg pellet).     

Activation of respiration in sea urchin spermatozoa by calcium ionophore A23187

The respiratory rate in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, in Na+-free seawater, where sperm are immotile and their respiration remains inactive, was stimulated by calcium ionophore A23187. Addition of ionophore A23187 to Na+-free seawater induced swimming as well as activating energy metabolism in sea urchin sperm. The increase of respiratory rate and the initiation of motility in sperm were independent of external Ca2+.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchins, Anthocidaris crassispina, Clypeaster japonicus and Pseudocentrotus depressus , decreases markedly in the presence of homologous unfertilized eggs fixed with glutaraldehyde. No decrease in the rate of respiration occurs in the presence of fixed fertilized eggs. Fixed unfertilized eggs of different sea urchin species do not cause any change in the rate of sperm respiration. Spermatozoa adhere only to the fixed unfertilized eggs of the same species and are removed by a stirring for 5 min on a magnetic stirrer. The spermatozoa thus removed, are immotile and their respiratory rate is quite lower than that of motile spermatozoa in a control suspension stirred for 5 min. Intact spermatozoa adhere to the fixed eggs, from which the attached spermatozoa have been removed, and the respiratory rate of the spermatozoa also becomes quite low.