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.     

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 : 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 : 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.     

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 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.     

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.     

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.     

Action of nitro- and halophenols upon oxygen consumption and phosphorylation by a cell-free particulate system from arbacia eggs

1. The ability of 4,6-dinitrocresol and eight other substituted phenols to stimulate oxygen uptake and inhibit phosphorylation by a cell-free particulate system from unfertilized Arbacia eggs has been determined. Five of those agents can produce both stimulation of oxygen consumption and inhibition of phosphorylation; one inhibits both oxygen consumption and phosphorylation; and two have no effect on either oxygen consumption or phosphorylation. In every case the effects of these substituted phenols upon the cell-free particulate systems parallel those upon oxygen consumption and cleavage in the intact fertilized Arbacia eggs. 2. The data suggest that energy for cleavage of the Arbacia egg is provided at least in part by oxidative phosphorylation and that substituted phenols may block cleavage by interfering with generation and transfer of high-energy phosphate groups.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VII. OBSERVATIONS ON THE AMOUNT AND POSSIBLE FUNCTION OF DIPHOSPHOTHIAMINE (COCARBOXYLASE) IN EGGS OF ARBACIA PUNCTULATA

1. Methods suitable for the determination of diphosphothiamine (cocarboxylase) in eggs of Arbacia punctulata have been developed. Quantitative extraction of the cocarboxylase was effected by combining the use of thiamine hydrochloride in the extraction fluid with critical adjustment of the pH of extraction to pH 6.3–6.7. 2. The unfertilized eggs were found to contain the equivalent of 2 to 3 micrograms of natural yeast cocarboxylase per gm. of wet eggs; the cocarboxylase content of the 30 minute and 10 hour fertilized eggs was somewhat less (Table III). 3. In preliminary experiments, Arbacia egg cytolysates were found to cause pyruvic acid to disappear. The rate of such disappearance was apparently greater under aerobic than under anaerobic conditions; it was also greater for cytolysates from fertilized eggs than for cytolysates from unfertilized eggs (Table IV).     

THE RELATION BETWEEN THE ELECTRICAL CONDUCTIVITY OF THE EXTERNAL MEDIUM AND THE RATE OF CELL DIVISION IN SEA URCHIN EGGS

Dilution of sea water with isotonic sugar solution leaves the rate of cleavage of Arbacia eggs almost unchanged until the proportion of sea water is decreased to 20 or 25 volumes per cent. From this point cleavage becomes progressively slower with further dilution. Many eggs fail to cleave at dilutions of 5 to 6 volumes per cent. No cleavage occurs in 2 volumes per cent sea water or in pure sugar solution. Eggs returned from these media to sea water resume cleavage and development. There is thus no relation between the rate of cleavage and the electrical conductivity of the medium, except possibly within the range of dilutions from 20 to 5 volumes per cent sea water. In this range cleavage rate decreases as conductivity decreases, but the relation is not a linear one.     

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).     

CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H³-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H³-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.     

Changes in the potassium content of sea urchin eggs on fertilization

In the eggs of Arbacia lixula and Paracentrotus lividus an uptake of K occurs during the first 10 minutes following fertilization. Between 10 and 40 minutes K is then released. Both in Arbacia and in Paracentrotus the minimum point of the curve coincides with the nuclear streak stage. A maximum loss of 25 per cent in Arbacia and 20 per cent in Paracentrotus with respect to the amount present in the unfertilized eggs has been found. From 40 minutes up to 1 hour K undergoes a further increase and when the first cleavage sets in the same amount of K is present as in the unfertilized eggs. By treating the eggs with K-free artificial sea water it has been established that about 60 per cent of the K content of the eggs is in a non-diffusible condition. Also under such conditions the eggs when fertilized are able to take up even the very small amount of K present in the medium that was released by them prior to fertilization.     

ELECTRIC IMPEDANCE OF SINGLE MARINE EGGS

Alternating current impedance measurements have been made on several single marine eggs over the frequency range from 1 to 2500 kilocycles per second. The eggs were placed in the center of a short capillary made by heating the end of a 2 mm. thin walled glass tube until it nearly closed, and electrodes were placed in the sea water on each side of the egg. When it is assumed that the membrane conductance is negligible, the membrane capacity and internal resistances of unfertilized and fertilized Arbacia eggs agree with the values obtained from suspensions. Preliminary data on centrifugally separated half Arbacia eggs, and whole Cumingia and Chaetopterus eggs are given.     

ACTION OF PAPAIN AND FICIN ON TADPOLES AND ARBACIA EGGS

Living tadpoles and Arbacia eggs are not digested by ficin or papain although the dead organisms are. Arbacia eggs develop in papain solutions but the cells become separated. Development is normal in ficin and trypsin solutions.     

ELECTRIC IMPEDANCE OF FERTILIZED ARBACIA EGG SUSPENSIONS

From the low frequency alternating current impedance and the volume concentrations of suspensions of Arbacia eggs, it is shown that the high resistance membrane is either at or very near the plasma membrane for both unfertilized and fertilized eggs, and that the specific resistances of the perivitelline space and fertilization membrane are not greatly different from that of sea water. The effect of the capacity element which appears after fertilization at intermediate frequencies is considerably less than in the earlier experiments on Arbacia and Hipponoë eggs. These findings indicate that the fertilization membrane does not have the high capacity previously attributed to it and that the increase in membrane capacity takes place at or near the plasma membrane.     

ON THE RATE OF OXYGEN CONSUMPTION BY FERTILIZED AND UNFERTILIZED EGGS : V. COMPARISONS AND INTERPRETATION

1. The rate of oxygen consumption by eggs may not merely undergo no change at fertilization, as in the case of the starfish, but it decreases to about half in Chaetopterus and in Cumingia. 2. The absolute rate of oxygen consumption in mm.³ O₂ per hour per 10 mm.³ eggs differs widely in several species of unfertilized eggs. It is very low in the sea urchin, intermediary in Nereis, and high in Chaetopterus and Cumingia. The range for these eggs is approximately 0.4 to 3.1 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C., in the ratio of about 1:8. 3. The absolute rates of oxygen consumption by the same fertilized eggs are much more nearly the same. They lie within the range 1.3 to 2.0 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C., in the ratio of approximately 1:1.5. Within this same range lie the values obtained by a number of investigators using a variety of eggs of invertebrates from several phyla. Amoeba proteus and frog skin also are within this range (see Fig. 2). 4. The changes in rate of oxygen consumption at fertilization by the different species of eggs, differing both in direction and magnitude, appear to be such as to bring the rate, when development is initiated, to about the same rate, which is also the rate of other comparable normally growing cells. 5. The direction and magnitude of the change in rate at fertilization therefore appears in the cases cited to be primarily a function of the absolute rate of oxygen consumption by the unfertilized eggs, which are characterized in their peculiar inhibited condition, among other things, by a wide range of respiratory rates. 6. It is not to be supposed that this range of rates will apply at all universally to eggs, especially to eggs of extremes in proportional content of inert materials, such as large yolky eggs. Fish and amphibian eggs for example respire at a much lower rate per unit volume. The effect on surface: volume ratios attending extremes of cell size might also be expected to shift the absolute rate. 7. The absolute rate of oxygen consumption by the eggs of the alga Fucus vesiculosus is considerably higher than the rates of the animal eggs measured. It is of the same order of magnitude as the rates of several other small-celled algae, which respire at a greater rate per unit volume than most non-motile animal cells. 8. The comparatively high rates of oxygen consumption by the inhibited (unfertilized) eggs of Chaetopterus and Cumingia are not directly associated with nuclear or morphological activity of the cell since they continue at the high rate for hours after cessation of the brief initial nuclear activity, which takes place when the eggs are placed in sea water. 9. It is concluded that the rate of oxygen consumption is not necessarily and probably not generally the limiting factor which causes inhibition of the unfertilized egg. Increase in rate of oxygen consumption is not directly related to the initiation of development, in general, nor even necessarily concomitant. It is not improbable that the low rate of oxygen consumption is an immediate part of the cause of inhibition of the unfertilized sea urchin egg, but this is a special case. 10. This thesis, that the rate of oxygen consumption is not necessarily nor ordinarily the limiting factor in the inhibition of the unfertilized egg, and conversely that increase in the rate of oxygen consumption is not usually the essential feature of fertilization, is quite in agreement with the general relations between the rate of oxygen consumption on the one hand and anesthesia, growth, and development on the other in fertilized eggs and other organisms. 11. This conclusion is opposed to Loeb''s explanation of the essential feature of fertilization, as an increase in oxidation rate or more strictly to generalization of his hypothesis to include eggs other than those of the sea urchins (or of other similar special cases which may be discovered). It extends to fertilization (the initiation of development) his and Wasteney''s well established conclusion that "oxidation is not the independent variable in development." 12. It is suggested that the crux of the problem of fertilization lies in the nature of the inhibition of the unfertilized egg. Certain similarities between this condition, arrived at spontaneously in the case of the egg cell, and the condition of cells in narcosis or anesthesia are pointed out. 13. Although the rate of oxygen consumption by the unfertilized eggs of Chaetopterus and Cumingia cannot be regarded as the limiting factor which causes the inhibition of the eggs, in these and other cases with different absolute rates, it appears highly probable that the rate of oxygen consumption is in some way, at present obscure, tied up with or related to the condition of inhibition. This seems probable especially in view of the sharp change in rate which in most cases immediately attends cessation of the inhibition, but the relationship may be a non-causal one, as in narcosis. 14. It must be borne in mind that oxygen consumption is not necessarily a complete measure of oxidation, and that other measures such as of heat and metabolite production are necessary before the complete amount of oxidation is known. When these are completely worked out, if free energy relations are known, it is probable that more direct and inclusive relations may be found between oxidation, growth, development, and anesthesia. Generalization of Loeb''s hypothesis, using "oxidation" in the broad sense might then turn out to hold, with fertilization fitting into the general scheme, but there is no basis for it at the present time.     

MACROMOLECULAR EVENTS LEADING TO CELL DIVISION IN TETRAHYMENA PYRIFORMIS AFTER REMOVAL AND REPLACEMENT OF REQUIRED PYRIMIDINES

Tetrahymena pyriformis were brought to a non-growing state by removal of pyrimidines from their growth medium. During pyrimidine deprivation cell number increased 3- to 4 fold, and this increase was accompanied by one or more complete cycles of macronuclear DNA replication. Autoradiographic studies show that endogenous protein and RNA were turning over throughout starvation and that RNA breakdown products were used to support the DNA synthesis that occurred during the early period of starvation. However, after 72 hours of starvation all DNA synthesis and cell division had ceased. Feulgen microspectrophotometry shows the macronuclei of these cells to have been stopped at a point prior to DNA replication (G1 stage). After pyrimidine replacement the incorporation of H³-uridine, H³-adenosine, and H³-leucine was measured by the autoradiographic grain counting method. The results indicate that RNA synthesis began to increase almost immediately, but that there was a lag of almost an hour before an increase in protein synthesis. In agreement with the autoradiographic data, chemical data also show that cellular content of RNA began to increase shortly after pyrimidine replacement but that cellular protein content did not increase until about one hour later. Pulse labeling of the cells with H³-thymidine at intervals after pyrimidine replacement shows that labeled macronuclei first began to appear at 150 minutes; that 98 per cent of the macronuclei were in DNA synthesis at 240 to 270 minutes; and that the percentage then began to decrease from 300 to 390 minutes, at which time only 25 per cent of the macronuclei were labeled. Cellular content of DNA did not increase for at least 135 minutes after pyrimidine replacement; however, just before the first cells divided (360 minutes) the DNA content had doubled. After pyrimidine replacement the cells first began to divide at 360 minutes, and 50 per cent had divided at 420 minutes; however, all cells had not divided until 573 minutes. This technique of chemical synchronization of cells in mass cultures makes feasible detailed biochemical analysis of events leading to nuclear DNA replication and cell division.     

THE EFFECT OF URETHANE ON THE CONSUMPTION OF OXYGEN AND THE RATE OF CELL DIVISION IN THE CILIATE TETRAHYMENA GELEII

1. The inhibition of oxygen consumption produced by a series of concentrations of ethyl carbamate has been measured in the protozoan Tetrahymena geleii. 2. The relation found between the narcotic concentration and its effect on respiration leads to the conclusion that urethane has two distinct modes of action in this cell. The respiratory data can be accurately predicted by assuming that the inhibitor acts on two independent parallel respiratory systems. 3. Complete suppression of cell division in this organism is brought about by approximately 0.1 M urethane. 4. Urethane concentrations up to 0.1 M affect primarily only one of the two postulated respiratory systems. The mechanism of the narcosis of cell division in this organism by urethane thus appears to be inhibition of this "activity" system.