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.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VIII. THE DIPHOSPHOPYRIDINE NUCLEOTIDE (COZYMASE) CONTENT OF EGGS OF ARBACIA PUNCTULATA

1. The diphosphopyridine nucleotide content of Arbacia eggs has been measured manometrically and found to be approximately 250–500 micrograms per gm. wet weight of eggs, the value varying with individual egg samples and with the state of development of the eggs. Of the total diphosphopyridine nucleotide present, approximately 25–40 per cent is in an alkali-stable, presumably the dihydro, form. 2. Tests for triosephosphate and glutamic acid dehydrogenases carried out on Arbacia egg cytolysates were negative.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VI. OBSERVATIONS ON THE GLYCOGEN CONTENT,CARBOHYDRATE CONSUMPTION,LACTIC ACID PRODUCTION,AND AMMONIA PRODUCTION OF EGGS OF ARBACIA PUNCTULATA

1. Under the present conditions of experiment, Arbacia eggs were found to contain an average of 110 mg. of acid-hydrolyzable carbohydrate (calculated as glucose) per gm. of egg protein. This carbohydrate was almost all in the egg proper, little or none being found in the jelly. To permit conversion of the data to other bases of reference the relation of nitrogen content to wet and dry weight and to egg number were determined. The eggs were found to contain 23.9 per cent solids, 0.10 mg. nitrogen per mg. dry weight, and 5.93 mg. nitrogen per 10⁶ cells. From these results, about 7 per cent of the egg dry weight is acid-hydrolyzable carbohydrate and about 65 per cent is protein. 2. Approximately one-half of the total acid-hydrolyzable carbohydrate was isolated in the form of an alkali-stable, alcohol-precipitable carbohydrate. This substance gave a typical glycogen color test with iodine, yielded glucose on acid hydrolysis, and had, within the limits of experimental error, the same optical rotation as glycogen from other animal sources. Since known amounts of glycogen were completely recovered when carried through the isolation process, the nature of one-half of the acid-hydrolyzable carbohydrate of Arbacia eggs remains undetermined. 3. In order to gain some estimate of the extent to which Arbacia eggs utilize their total carbohydrate for development, determinations of the oxygen consumption, respiratory quotient, carbohydrate consumption, lactic acid production, and ammonia production were made. While all samples of eggs were found to utilize carbohydrate from the 15th to the 24th hours of development at 20°C., certain samples of eggs consumed little or no carbohydrate from the 1st to the 6th hours, the period during which cell division proceeds most rapidly. In a number of instances where carbohydrate breakdown was lacking, a substantial proportion of the oxygen consumption could be accounted for on the basis of processes involving oxidation of protein or protein breakdown products.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

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.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

FERTILIZATION AND THE TEMPERATURE COEFFICIENTS OF OXYGEN CONSUMPTION IN EGGS OF ARBACIA PUNCTULATA

The eggs of A. punctulata have a high temperature coefficient in the resting state: Q ₁₀ = 4.1. On fertilization and on cytolysis the temperature coefficient falls to less than half the resting value: Q ₁₀ = 1.8 and 1.9 respectively. The factor by which oxygen consumption increases on fertilization is a variable, its magnitude depending on temperature as well as on egg species. It is nearly ten times greater at 11°C. and only double at 29.9°C. By extrapolating to 32°C. there would be no increase on fertilization. Critical thermal increments common to many oxidations, 6,500, 10,800, and 12,500, have been found. The possible significance of these results is discussed in relation to the catalytic mechanisms and structural organization of the egg cell.     

THE OSMOTIC PROPERTIES OF LIVING CELLS (EGGS OF ARBACIA PUNCTULATA)

We have attempted to answer the question: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight.     

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.     

REINSEMINATION OF FERTILIZED SEA URCHIN (ARBACIA PUNCTULATA) EGGS

Embryos of Arbacia punctulata , treated to remove the fertilization membrane and hyaline layer, were mixed with sperm (reinseminated) at 20–30 min (streak stage) and at 70 min (2-cell stage) postinsemination. Sperm, incorporated into embryos reinseminated at the streak stage, metamorphosed into male pronuclei which subsequently migrated to and fused with the zygote nucleus. Although blastomeres were capable of incorporating sperm and forming fertilization cones, less than 10% of the 2-cell stage embryos reinseminated. Sperm, were found entrapped within an amorphous material along the surface of 2-cell stage embryos; many had failed to undergo an acrosome reaction. These results indicate that conditions necessary for incorporation and metamorphosis of sperm nuclei into male pronuclei are present in the embryo after the normal period of fertilization.     

ON THE RATE OF OXYGEN CONSUMPTION BY FERTILIZED AND UNFERTILIZED EGGS : IV. CHAETOPTERHS AND ARBACIA PUNCTULATA

1. Unfertilized eggs of Chaetopterus consume about 2.4 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 2. In the 1st hour after fertilization, the fertilized eggs consume oxygen at about 53 or 54 per cent of this rate, which is about 1.3 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 3. For the first 6 hours after fertilization, at 21°C., the curve of the rate of oxygen consumption is slightly asymmetrically sigmoid. The prefertilization rate is regained between 4½ and 5 hours after fertilization. Soon after 6 hours, ciliary activity begins, and the rate of oxygen consumption rises rapidly. 4. The unfertilized eggs of Arbacia punctulata consume about 0.36–0.5 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. The absolute determination is difficult as these eggs are highly sensitive to shaking in the manometer vessels, and these difficulties are discussed. 5. The fertilized eggs of Arbacia punctulata consume oxygen at the rate of about 2.0 mm.³ O₂ per hour per 10 mm.³ 21°C. At 1 hour after fertilization the rate is already rising. 6. A comparison of the absolute rates of oxygen consumption, and the changes in rate at fertilization of these and a number of other eggs, together with a theoretical discussion, and a discussion of discrepancies in measurements on the eggs of Arbacia punctulata, is contained in the fifth paper of this series (21).     

A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA

A fine structural study has been carried out on the various formed elements present before, during, and after the first cleavage division, not only in normally developing Arbacia eggs, but also in eggs which have been induced to cleave prematurely by high-pressure centrifugation. The aim has been to ascertain whether or not any of the morphologically identifiable components may be involved in initiating the furrowing process. Also, attention has been given to the fine structure of the cytoplasmic cortex, particulary in the walls of the furrow, in the hope of reaching a better understanding of the mechanics of cleavage. The annulate lamellae and the membranous envelope of the nucleus are the only formed elements which disappear shortly before cleavage, not only in eggs undergoing normal division, but also in eggs which have been induced to cleave ahead of schedule by high-pressure, high-force centrifugation. Therefore, it is suggested as a tentative hypothesis that materials liberated upon disintegration of the nuclear membrane and the annulate lamellae play an essential role in initiating and effecting the furrowing reaction, especially since the stratification of these elements in experimentally induced eggs corresponds to the position of the developing furrow. Another of the membranous elements in the egg, the Golgi complex, shows considerable modification as a result of high-pressure centrifugation, but these structures do not undergo disintegration. Rather, they become curled into rounded bodies. The vacuole population is not greatly affected by inducing treatments. During cleavage, both naturally occurring and experimentally induced, a considerable number of 50 A filaments appear in the denser cytoplasmic cortex, but only in the walls of the furrow. These filaments are similar to those which have been demonstrated in a number of contractile cells. Accordingly, it is suggested that this fibrillar system may be actively involved in the development of the cleavage force.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : II. STIMULATION OF CELLULAR OXIDATION AND REVERSIBLE INHIBITION OF CELL DIVISION BY DIHALO AND TRIHALOPHENOLS

The dihalo and trihalophenols, and phenols containing both halo and nitro substituents in the same molecule, produce, in fertilized eggs of Arbacia punctulata, a rise in rate of oxygen consumption and a reversible block to cell division. To define the conditions which affect the degree of this activity, the following factors have been varied: the arrangement of substituents in the molecule, the concentration of reagent, and the time after fertilization at which the reagent is added. The stimulation of oxygen consumption and reversible block to cell division produced by the dihalophenols are qualitatively the same as those previously produced in fertilized Arbacia eggs by certain dinitrophenols. To yield optimum respiratory effect and maximum division block, it usually requires a higher concentration of dihalo than of the corresponding dinitrophenol. For example, with fertilized Arbacia eggs at 20°C. 2,4-dinitrophenol, in optimum concentration of 3 x 10^–5 molar, raises oxygen consumption to 292 per cent of normal (4). The corresponding values for two dihalo analogues are: 2,4-dichlorophenol, 10^–4 molar and 236 per cent; 2,4-dibromophenol, 6 x 10^–5 molar and 282 per cent. The halophenols differ from the nitrophenols in two interesting respects: (a) The monohalophenols produce little or no oxidative stimulation or division block in fertilized Arbacia eggs; p-nitrophenol is very active in both respects. (b) The symmetrical trihalophenols have an appreciable ability to stimulate oxygen consumption and block division; symmetrical trinitrophenol is inactive in both respects (4). The increases in oxygen consumption produced in fertilized Arbacia eggs by 2,4-dichloro and 2,4-dinitrophenol are larger than the percentage increases given by methylene blue and o-cresol indophenol under the same experimental conditions. The dihalo and dinitrophenols produce a reversible block to the cell division of fertilized marine eggs. The oxidation-reduction indicators, in contrast to the dihalo and dinitrophenols, block cell division irreversibly and fertilized eggs of Arbacia do not recover from optimum respiratory stimulating concentrations of these oxidation-reduction dyes. The present experiments with halophenols are in harmony with and lend considerable support to the hypothesis (4) that nitro and similarly substituted phenols derive their biological activity from the presence and properties of the phenolic OH group, as modified by proper substitution in the phenolic benzene ring.     

INCREASED PERMEABILITY TO WATER OF AGING UNFERTILIZED EGGS (ARBACIA PUNCTULATA)

1. Unfertilized Arbacia eggs, subjected to 50, 60, or 80 per cent sea water, at the same temperature, pH, and duration, swelled progressively faster with age. Swelling increased 12.2 to 39.6 per cent. 2. Permeability to water was calculated by the method of Lucké and McCutcheon. For freshly shed eggs from nine different females, permeability varied from 0.078 to 0.124, or 59 per cent. This variation was not due to differences in age nor to physiologic condition but was correlated with size of eggs. 3. About twenty eggs were tested each age to 60 hours after shedding. The permeability rate increased with age in all nine experiments. 4. The total increase ranged from 30 to 69 per cent. 5. The increase was slow but constant or almost constant during early and intermediate ages. 6. The increase began at earliest ages. 7. An increase may not be demonstrable for any short interval but is readily demonstrable for longer intervals (6 or more hours), or, from the entire curve. 8. Permeability increased faster at late ages. For special factors during late ages see text. 9. Injury by experimental procedure was not induced until ca. the 24th hour. Injury by aging was not detectable during the first 6 to 9 hours. It increased progressively between 10 and 24 hours, and very rapidly thereafter. Permeability increased at approximately the same rate during early and intermediate ages. Permeability therefore was not correlated with degree of injury. 10. The troublesome factor in the permeability equation was initial size, or V_o. Several methods were used to obtain this V_o, viz. (a) extrapolations approximating known cyclical change in size, (b) extrapolations approximating control sizes, (c) constant or average of all V_o''s, (d) maximum V_o approximating control V_o''s. Recalculation with these different V_o''s gave approximately the same permeabilities. 11. The increase in permeability with age was substantially the same whether "approved," or other possible rates or average rates or range of rates be used as criteria of change. 12. When the data were recalculated by integrating the equation of Northrop, the increases in permeability with age were essentially the same as the increases calculated by the graphic method of Lucké.     

THE PYRUVATE METABOLISM OF SEA URCHIN EGGS DURING THE PROCESS OF CELL DIVISION

The eggs of Arbacia and starfish contained about 70 and 25 micrograms of pyruvate per gm. of dry cells respectively. Arbacia eggs utilized added pyruvate, although the O₂ uptake did not increase. On fertilization the utilization of pyruvate increased sevenfold. This pyruvate seems to be metabolized, as in other cells, with diphosphothiamine as coenzyme. The diphosphothiamine content of fertilized and non-fertilized eggs was about 16 micrograms; that of sperm, 30 micrograms. Penetration of sperm into the egg and fertilization with cell division to the pluteus stage did not bring forth appearance of succino-dehydrogenase. The possible mechanism of fertilization and cell division is discussed.     

THE EFFECTS OF NICOTINE ON FERTILIZATION IN THE SEA URCHIN, ARBACIA PUNCTULATA

The number of sperm incorporated into eggs made polyspermic with varying concentrations of nicotine (0.025–0.25%, v/v) appears to be directly related to the concentrations employed. The cortical response is morphologically equivalent to that observed in control preparations. Shortly after their incorporation all of the spermatozoa undergo structural events normally associated with the development of the male pronucleus in monospermic eggs. During the reorganization of the spermatozoa, sperm asters are formed. The number of male pronuclei that initially migrate to and encounter the female pronucleus is usually one to three. When pronuclei come into proximity to one another the surface of the female pronucleus proximal to the advancing male pronuclei flattens and becomes highly convoluted. Subsequently, the pronuclei contact each other and the outer and inner membranes of the pronuclear envelopes fuse, thereby producing the zygote nucleus. The male pronuclei remaining in the zygote after this initial series of pronuclear fusions continue to differentiate, i.e. they enlarge, form nucleolus-like bodies, and undergo further chromatin dispersion. In approximately 90% of the zygotes, all of the remaining male pronuclei progressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 min postinsemination. Mitosis and cleavage of the polyspermic zygote occurs later than in monospermic eggs.     

FURTHER STUDIES ON CELL DIVISION WITHOUT MITOTIC APPARATUS IN SEA URCHIN EGGS

A large quantity of paraffin oil, sucrose solution, or sea water was injected into the eggs of the heart urchin Clypeaster japonicus shortly before the onset of the first cleavage. The injected oil became spherical, pushing the mitotic apparatus aside. The sucrose solution mixed with the protoplasm and caused disintegration of the mitotic apparatus, and the sea water formed a vacuole at the center of the cell. In all these cases, cleavage may take place almost normally in spite of the absence of the mitotic apparatus or its displacement within the cell. In some eggs, furrowing may take place when more than fifty per cent of the endoplasm has been replaced with sea water before onset of cleavage.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : IV. COMBINED ACTION OF SUBSTITUTED PHENOLS,CYANIDE, CARBON MONOXIDE,AND OTHER RESPIRATORY INHIBITORS ON RESPIRATION AND CELL DIVISION

The effects of 4,6-dinitro-o-cresol and 2,4,5-trichlorophenol on the respiration and cell division of fertilized eggs of Arbacia punctulata have been determined in the presence of each of a number of respiratory inhibitors. The experimental results obtained appear to afford some understanding of the mechanism of action of the substituted phenols on respiration and on cell division. 1. From the fact that the stimulated respiration is completely cyanide and carbon monoxide sensitive, it may be concluded that all of the extra oxygen uptake induced in Arbacia eggs by 4,6-dinitro-o-cresol passes through the metal containing oxidase system. All of the extra oxygen uptake also passes through oxidative steps which can be poisoned by non-stimulating phenols like 2,4-dinitrothymol and 4-nitrocarvacrol, by phenylurethane, by 5-isoamyl-5-ethyl barbituric acid, by malonic acid, or by iodoacetic acid. To abolish all respiratory stimulation by suboptimum concentrations of 4,6-dinitro-o-cresol, each of these inhibitors must be present in a concentration which reduces the normal respiration in the absence of substituted phenols by at least 20–40 per cent. 2. The degree of reduction of the stimulated respiration by a given concentration of carbon monoxide or potassium cyanide depends on the concentration of 4,6-dinitro-o-cresol or 2,4,5-trichlorophenol, being most marked in suboptimum concentrations and least marked in greater than optimum concentrations of the substituted phenol. In contrast to this result, the reduction of the stimulated respiration by a given concentration of 5-isoamyl-5-ethyl barbituric acid or malonic acid is least marked in suboptimum concentrations and most marked in greater than optimum concentrations of the substituted phenol. 3. The present experiments appear to indicate that the inhibition of cell division by substituted phenols is not attributable to a direct action of these agents on mitotic processes nor to an overstimulation of any respiratory process. The inhibition of cell division appears to be associated with the inhibition, by the substituted phenols, of some component of the cyanide sensitive respiratory system. This inhibition is of such a type as to allow the overall respiration to proceed at a rate in excess of the control value, even when division is completely suppressed. The dependence of the division mechanism on a respiratory step which is relatively hypersensitive to poisoning by the substituted phenols is comparable to the dependence of the Pasteur reaction in certain normal and tumor tissues on an oxidative step which is specifically poisoned by the substituted phenols (16). The substituted phenols have no inhibiting effect in vitro on the principal metal containing respiratory catalysts or the principal dehydrogenases; they also do not inhibit the fermentative reactions involved in the anaerobic glycolysis of fertilized Arbacia eggs. It is therefore suggested that the respiratory inhibiting and division inhibiting effects of the substituted phenols may be attributable to the action of these substances on one or more of the oxidation-reduction or phosphorylating steps which are involved in the transfer of hydrogen from the dehydrogenase systems to the specifically cyanide sensitive oxidase mechanism of the eggs. The identification of the respiratory step poisoned by the substituted phenol would constitute an interesting contribution to the chemistry of cell division and experiments to this end are now in progress.     

A CYTOLOGICAL STUDY OF THE CENTRIFUGED WHOLE, HALF, AND QUARTER EGGS OF THE SEA URCHIN ARBACIA PUNCTULATA

While the ooplasmic components of centrifuged eggs of Arbacia punctulata do not stratify in homogeneous layers, we have obtained the following strata beginning with the centripetal end: lipid droplets, pronucleus, clear zone, mitochondria, yolk, and pigment. Whereas mitochondria may be found mingled with yolk bodies, we have never observed lipid droplets nor pigment bodies among any of the other inclusions. The so-called clear zone contains a heterogeneous population of inclusions: annulate lamellae, heavy bodies, Golgi complexes, and rod-containing vacuoles. The peripheral cortical granules of immature (germinal vesicle stage) and of mature eggs are not dislodged from the cortical ooplasm with the centrifugal force utilized. When the eggs are treated with urethane, prior to centrifugation, the cortical granules of mature eggs abandon their peripheral position. Further centrifugation of the initially stratified eggs produces nucleated and nonnucleated halves and the centrifugation of the halves results in quarters. The cytology of the halves and quarters is discussed. The halves and quarters have been activated with either sperm or hypertonic sea water. With the exception of the nucleated halves, we were unable to obtain plutei larvae from the other fractions (red halves and quarters). We believe that the lack of development of the various fragments is a function of the balance of particular inclusions necessary for differentiation.