THE METABOLISM OF Cl402 DURING AMPHIBIAN DEVELOPMENT

BY STANLEY COHEN*

(From the Edward Mallinckrodt Institute of Radiology, Washington University

School of Medicine, St. Louis, Missouri)

(Received for publication, May 10, 1954)

These exploratory studies were made to gain some insight into the chemical transformations which occur during early embryonic develop- ment. Eggs and embryos of the frog, Rana pipiens, were chosen because of the many studies on the embryology of this species and because of the availability of the material.

Previous work from other laboratories had indicated that intact frog embryos can take up from the medium only negligible amounts of radio- active inorganic phosphate (I), glycine (a), or methionine (3). Kutsky (1) succeeded in labeling frog’s eggs with P32 by injecting the adult female with radioactive inorganic phosphate at the time ovulation was induced. Friedberg and Eakin (2) and Eakin, Kutsky, and Berg (3) obtained a slight uptake of radioactive glycine and methionine after incubating sec- tioned embryos in a medium containing the amino acids.

In the present studies, it is shown that in slightly acid solutions (pH 6.4) a sufficient amount of C1402 is metabolized by the intact frog egg and embryo to permit chemical fractionation and identification of the radio- active compounds present at various stages of development.

Materials and Methods

Preparation of Eggs and Embryos-Frogs (Rana pipiens) were obtained from Wisconsin and ovulation was induced by the injection of pituitary glands. 36 hours after injection the eggs were stripped, fertilized, divided into groups of ten to twenty eggs each, and allowed to develop to the desired stage at room temperature (23-26’). The procedures followed are given in Hamburger’s manual (4). The development of the embryos was allowed to take place in a modified Holtfreter’s solution containing, per liter, NaCl 350 mg., KC1 5 mg., and CaClz 10 mg.

Incubation of Eggs and Embryos with Labeled C02-The stages selected for study were the unfertilized egg (stage I), mid-cleavage (stage 8), mid-

* Postdoctoral Fellow of the American Cancer Society. Present address, Depart- ment of Zoology, Washington University, St. Louis, Missouri. A preliminary report was presented at the meeting of the Federation of American Societies for Experi- mental Biology at Chicago, April, 1953 (Federation Proc., 12, 191 (1953)).

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338 METABOLISM OF d402 IN AMPHIBIA

gastrula (stage II), and neural fold (stage 14). The identification of the stages was made on the basis of the data provided by Shumway (5). 1200 eggs at the desired stage of development were placed in a 70 ml. cylinder containing 25 ml. of the modified Holtfreter’s solution, noted above, to which had been added Na2HP04 (final concentration 1OW’ M) and from 120 to 140 ,uc. of Cl4 as NaHC03. (Labeled NaHC03 was prepared from labeled BaC03 (15.1 to 26.7 mg. per mc.) as supplied by the Isotopes Division, Atomic Energy Commission, Oak Ridge, Tennessee.) 2 drops of indicator (bromocresol purple, 0.01 per cent) were added, and the cylin- der was flushed with oxygen and closed with a rubber stopper. Sufficient hydrochloric acid (0.1 M) was then injected through the rubber stopper to adjust the pH to 6.4. The vessel was then incubated for 5 hours at 25-26” with gentle shaking. When unfertilized eggs were used, the incubation was started 1 hour after the eggs were stripped to allow time for the jelly to swell. The volume of 1200 eggs varied from 29 to 37 ml. At the end of the incubation period, the vessel was chilled in an ice bath, sufficient NaOH (0.5 M) was injected to render the mixture alkaline to phenolphtha- lein, and the eggs were removed by filtration through a Biichner funnel.

Fractionation of Eggs-The eggs, after filtration, were transferred into chilled methanol (1 volume of eggs, with jelly, to 2 volumes of methanol), and homogenized in an Osterizer. The mixture was then acidified with a few drops of formic acid (until the protein coagulated), and the radioactive carbonate was removed by bubbling unlabeled carbon dioxide through the mixture. The mixture was then centrifuged and the residue washed twice with 50 ml. portions of an acidified methanol solution (0.1 ml. of formic acid per 100 ml. of 70 per cent methanol). This methanol fraction con- stituted the “soluble fraction.” The residue was treated with 20 ml. of an alcohol-ether mixture (3 : 1) and refluxed for 1 hour to remove the “lipide fraction.” This extraction was repeated three times. The lipide-free residue was then dried in air and extracted three times with 20 ml. portions of a hot 5 per cent trichloroacetic acid solution (15 minutes at 90”) to remove the “nucleic acid fraction” (6). The combined extract was then boiled to decompose the trichloroacetic acid. The final residue, the “protein fraction,” was washed with alcohol and ether and dried.

Assay of Radiocarbon-Aliquots of the above fractions were transferred to tared stainless steel dishes (2.8 sq. cm. area), and evaporated to dryness under infra-red lamps. The dishes were then assayed in a standard posi- tion under a thin window Geiger-Miiller counter. A counting efficiency of approximately 2.6 per cent was obtained, Corrections for self-absorption were made when necessary. Unless otherwise noted in the text, sufficient counts were made to reduce the statistical error to less than 5 per cent.

Paper Chromatography and Radioautography-Two-dimensional de-

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S. COHEN 339

scending paper chromatography was carried out with the solvent pairs, propanol-ammonia-mater (6: 3 : 1) and the upper phase of a tert-amyl alcohol-water-formic acid (3 :3: 1) mixture, with the procedures of Hanes and Isherwood (7). Before adequate chromatograms could be obtained, further treatment of the crude fractions was necessary.

The soluble fraction was evaporated to dryness under a stream of dry air, which finally was bubbled through a solution of NaOH. Only a negligible quantity of Cl4 could be detected in the NaOH solution. The residue was taken up in a few ml. of water and acidified to pH 2 to 3 with HCl and centrifuged to remove the small amount of insoluble material. The supernatant solution was extracted continuously for 18 hours with ether. The ether extract was evaporated to dryness, 2 ml. of water were added, and aliquots were chromatographed. The aqueous fraction was neutralized and evaporated to dryness. Since this material gave smears when attempts were made to chromatograph it, the dried residue was triturated with two 20 ml. portions of 90 per cent methanol and centrifuged, and the combined supernatant fluids were evaporated to dryness. 2 ml. of water were added to each fraction, the mixture was centrifuged, and aliquots of the supernatant fluid were chromatographed.

The nucleic acid fraction was evaporated to dryness and hydrolyzed for 1 hour at 100” with 3 ml. of 70 per cent perchloric acid according to the procedure of Marshak and Vogel (8) for the liberation of the free purines and pyrimidines. KOH (4 N) then was added to the hydrolysate to a final pH of 1 to 2. The mixture was centrifuged and the residue washed several times with water. Aliquots of the supernatant fluid were chro- matographed.

The protein fraction was hydrolyzed with 6 N HCl in an autoclave for 15 hours. The hydrolysate was taken to dryness several times in a vacuum desiccator over calcium chloride and potassium hydroxide and chromato- graphed.

After development of the chromatogram, the papers were radioauto- graphed in contact with Eastman Kodak x-ray film. Aliquots assaying about 2000 c.p.m. in the dish were exposed for 2 days. Other aliquots were exposed so that the product of the activity and the time of exposure remained approximately constant.

The radioactive regions thus outlined were compared with the positions of the various compounds as made visible by ultraviolet absorption (for the purines and pyrimidines) or chemical treatment. Amino acids were developed with a spray of 0.1 per cent ninhydrin in n-butanol, the paper being dried at 65”. Organic acids were developed with 0.04 per cent aqueous bromocresol purple solution after removing the formic acid solvent by autoclaving the paper for 20 minutes. Other reagents are noted in the

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340 METABOLISM OF Cl402 IN AMPHIBIA

text. In duplicate runs, the radioactive material was eluted with water and carrier was added, and the mixture was subjected to two-dimensional chro- matography. Identity was assumed when the carrier spot corresponded precisely with the location of a radioactive spot.

Degradation of Uracil-The following procedure was devised for the enzymatic degradation of 0.1 pmole quantities of uracil. Use was made of the enzymes uracil oxidase and barbiturase, discovered in a strain of Mycobacterium by Hayaishi and Kornberg (9). In principle, the method depends upon the oxidation of uracil to barbituric acid, the hydrolysis of barbituric acid to urea and malonic acid, and the hydrolysis of urea to ammonia and carbon dioxide. The carbon dioxide, coming from the C-2 position of uracil, is collected as BaC03 and counted.

Enzyme Preparations-Uracil oxidase was prepared by growing the Mycobacteriuml on a medium containing uracil (0.1 per cent), thymine (0.1 per cent), NaCl (0.08 per cent), KHzP04 (0.04 per cent), and MgS04.7HZ0 (0.02 per cent). The culture was maintained for 48 hours at about 26” with constant agitation. The cells were harvested by centrifugation. The yield of cells was approximately 1 gm. (wet weight) per liter of medium. The cells were ground with alumina as described by Hayaishi and Korn- berg, the paste being extracted with 5 volumes of glycylglycine buffer (0.02 M, pH 9.0) and centrifuged at 0” at 16,000 X g for 10 minutes. The supernatant solution could be stored for at least several weeks in the frozen condition,

Barbiturase was prepared in a similar manner with the following modifi- cations. In place of the thymine, glucose (0.2 per cent) was added to the medium. After 48 hours of growth, the cell extract was prepared by grinding with alumina and extracting with 5 volumes of phosphate buffer (0.02 M, pH 6.7). After centrifugation, the supernatant solution could also be stored for at least several weeks in the frozen condition.

Procedure-A solution containing approximately 0.1 pmole of uracil, the activity of which had been determined as an infinitely thin film, was evaporated to dryness in the outside chamber of a Conway microdiffusion dish under an infra-red lamp. 0.2 ml. of glycylglycine buffer (0.25 M, pH 8.9), 0.02 ml. of methylene blue (2.67 X 1W M), and 0.3 ml. of the uracil oxidase were added. In the central chamber was placed a tared steel counting dish containing a solution of Ba(OH)z in slight excess of the ex- pected quantity of CO?, and a drop of phenolphthalein indicator. The unit was sealed and incubated at 26” with gentle agitation for 1 hour. The amount of enzyme added was sufficient to oxidize over 95 per cent of the uracil present. in 30 minute s as determined spectrophotometrically

‘The strain of Mycobacterium was obtainedfrom Dr. Hayaishi, towhom the author is also indebted for helpful discussions.

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S. COHEN 341

(9). At the end of the incubation period the unit was opened slightly, and 4 mg. of urease, 0.1 ml. of glycylglycine buffer (0.5 M, pH 8.9), 5 @moles of sodium barbiturate, and 1 ml. of the barbiturase preparation were added quickly. The unit was again sealed and allowed to incubate for 1.5 hours. The quantity of enzyme used was sufficient to degrade over 95 per cent of the added barbituric acid in 45 minutes as determined spectrophotomet- rically (9). When the incubation was completed, excess 0.2 N H,S04 was added to the outer chamber, and the liberated CO2 was allowed to diffuse into the central dish for several hours. The barium carbonate was then dried under an infra-red lamp and counted, corrections being made for self-absorption.

It is advisable to duplicate the entire procedure by degrading a known sample of C-2-labeled uracil as a control in parallel with the unknown sample.

Distribution of Cl4 in Soluble, Lipicle, Nucleic Acid, and Protein Fractions of Developing Embryo-The percentage distribution of the Cl402 metab- olized during the 5 hour incubation period by the unfertilized egg, the blastula, the gastrula, and the neurula is shown in Table I.

There was no consistent change in the dry weight of the lipide and protein fractions at the various stages. The lipide content of all stages examined varied between 320 and 400 mg. per 1200 eggs, and the protein content varied from 880 to 1020 mg. per 1200 eggs. These data are con- sistent with those reviewed by Needham (lo), who noted that, in Rana temporaria, approximately 60 per cent of the dry weight of the eggs was protein and 21 per cent lipide. Practically no fat disappears from the amphibian egg before hatching. Gregg and Ballentine (11) reported that the total nitrogen content of Rana pipiens embryos remained nearly constant throughout development. The average total nitrogen of the jelly-free embryo at the beginning of development was 162 y, of which approximately 4 y resided in the total non-protein nitrogen fraction.

From 2 to 5 per cent of the radioactivity offered was incorporated by the embryos. The absolute amounts taken up at each stage could not be calculated from the data obtained for the following reasons. The carbon- ate content of the embryos varied considerably at different stages (12) and was not determined in these exploratory experiments. In addition, the carbon dioxide produced by the nenrula during the 5 hour incubation was much greater than that produced by the unfertilized egg (12). Fi- nally, although the Cl4 content of the incubation mixture was held approxi- mately constant, the amounts of carbonate added varied by a factor of 2. However, some approximations could be made. In one experiment at the neurula stage, the specific activity of the carbonate remaining in the medium after completion of the incubation was determined and found to

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342 METABOLISM OF Cl402 IN AMPHIBIA

be 0.09 X lo6 c.p.m. per mg. of BaC03. The initial specific activity of the added carbonate was 2.38 X lo6 c.p.m. per mg. in a total quantity of 3.5 mg. Thus the added carbonate was diluted approximately 26-fold, and about 90 mg. (as BaC03) were contributed to the medium by the carbonate present in the jelly and the embryo, and produced by respira- tory activity. If it is assumed that this added carbonate was contributed to the medium at a uniform rate throughout the incubation, then the average specific activity of the medium was 0.31 X lo6 c.p.m. per mg. of

TABLE I

Per Cent Distribution of Cl4 during Amphibian Development

The number of experiments at each stage is indicated in parentheses. The total counts incorporated were calculated by summing the counts in the four fractions isolated. All the counts were corrected for self-absorption.

Fraction

Soluble. Lipide Nucleic acid. Protein

Total c.p.m. incorpo- rated..

Total initial c.p.m. in medium*.

I

-

T

Stage of development

Unfertilized (3) Blastula (3) Gastrula (2) Neurola (2)

per cent per cent per cent per cent

71 -75 45 -54 32 -41 16 -21 O.l- 0.4 0.5- 0.7 0.9- 1.2 0.3- 0.6

21 -25 33 -48 45 -52 53 -58 3 -4 7 -13 14 -15 21 -32

..6-2.5 X 1052.3-3.0 X 1052.3-2.8 X 1053.5-4.4 X lo5

6.8-8.3 X lo6

* The added radioactive carbonate varied from 2 to 4 mg. as BaC03 per vessel.

BaC03. Since 0.35 X lo6 c.p.m. were recovered in the eggs, approximately 1.1 mg. of carbonate (as BaC03) were fixed by the 1200 neurula.

It appeared probable that only the uncharged molecules of dissolved carbon dioxide could penetrate the egg membranes since, in preliminary experiments run at a pH of 9, almost no radioactive carbonate was incor- porated. Similar results have been obtained with sea-urchin eggs (13).

In several experiments, after the incubation period was over some of the eggs were allowed to hatch and were normal in appearance.

Radioactive Components of Soluble Fraction-The nature of the com- pounds present in the soluble fraction was examined by the chromato- graphic and radioautographic techniques. In Fig. 1 are shown diagram- matically the positions on the two-dimensional chromatogram of all the active materials found in the soluble fractions of the developing embryo.

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S. COHEN 343

Radioactive aspartic, glutamic, malic, citric, fumaric, succinic, and ureido- succinic acids were identified. An insufficient amount of the ureidosuccinic acid was present to respond to the p-dimethylaminobenzaldehyde reagent for the ureido group (14). It was further identified by eluting the material, and converting it to 5-acetic acid hydantoin by adding 5 ml. of 6 N HCl and heating almost to dryness (15). After repeating the acid treatment three times, the material was chromatographed with an authentic sample of &acetic acid hydantoinz and again the radioactive material coincided with the carrier added.

Hydantoin derivatives may be rendered visual on paper chromatograms by spraying the paper with the ninhydrin reagent, heating it for 15 minutes

T }RF=O.3

ACIDIC SOLVENT +

FIG. 1. Diagram of the positions on the chromatogram of the radioactive com- ponents in the soluble fraction of the developing frog embryo. The following abbre- viations are used: aspartic acid, A; glutamic acid, G; malic acid, M; succinic acid, S; fumaric acid, F; citric acid, C; ureidosuccinic acid, U. The dotted circles indicate the positions of unidentified compounds. The chromatogram was developed first with the basic solvent (ammonia-propanol-water) and then with the acidic solvent (formic acid-amyl alcohol-water).

at 70-80”, and finally spraying with a 5 per cent ammoniacal silver nitrate solution. The whole paper immediately turns brown except for the hy- dantoin area, which remains colorless. Pyrimidine derivatives also re- spond to this reagent.

The percentage distributions of Cl4 in the various compounds isolated were determined by elution of the spots with three 5 ml. portions of hot water, the extracts being plated and counted directly. The activity was then compared to the original activity of the aliquot placed on the paper. Since from only 70 to 82 per cent of the activity could be accounted for in this manner, the results, shown in Table II, are approximations.

The following changes in the isotope concentrations of the compounds

2The writer wishes to thank Dr. Irving Lieberman for the sample of 5-acetic acid hydantoin.

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344 METABOLISM OF Cl402 IN AMPHIBIA

isolated at the various stages appeared to be significant. Aspartic acid, which accounted for almost 60 per cent of the isotope in the unfertilized egg, accounted for only about 12 per cent in the blastula and 18 per cent in the gastrula, and then increased again to about 38 per cent in the neu- rula. This decrease and subsequent increase in activity were paralleled by a decrease and increase in the relative amount of aspartic acid present as determined by visual examination of the intensity of the ninhydrin spots. Concomitant with the drop in the activity of the aspartic acid, there was a sharp rise in the amount of the isotope in unknown Compound 1, which increased from 0.3 per cent in unfertilized eggs to 14 per cent in

TABLE II

Per Cent Distribution of Cl4 in Soluble Fraction

Compound

Aspartic acid. Glutamic “ Malic “ Succinic “ Fumaric ” Citric “ Ureidosuccinic acid. Unknown Compound 1.. Other unknowns.

Per cent recovery.

Unfertilized Blast& Gastrula

peu cent per cent per cent 58 12 18 13 28 20

2 2 5 0.6 1 4 0.5 0.5 0.6 * 3 1 * 3 5

0.3 14 11 4 6 7

78 70 72

Stage of development

* The compound was not detectable in the aliquots used.

N-3lda

per cent

38 22

4 4 1 2 1 *

10

82

the blastula and 11 per cent in the gastrula but which could not be detected in the neurula. Citric and ureidosuccinic acids, which could not be de- tected in the unfertilized eggs, were detectable in later stages.

The data presented in Table II were obtained from single experiments at each stage. Visual examination of the radioautographs from duplicate experiments indicated a very similar distribution to that presented above.

With respect to the unknown compounds indicated in Fig. 1, the follow- ing may be said. Unknown Compound 1 was found both in the ether extract (as were the acids of the tricarboxylic acid cycle) and in the 90 per cent methanol-soluble fraction. The compound failed to react with the following spray reagents: bromocresol purple for acids, ninhydrin for amino acids, 2,4-dinitrophenylhydrazine for keto groups, p-dimethyl- aminobenzaldehyde for ureido derivatives, Folin-Wu reagents for reducing

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S. COHEN 345

sugars, Pauly reaction for imidazoles, Sakaguchi reaction for guanidino derivatives, and ammoniacal silver nitrate for dihydroxy compounds. There was no detectable absorption or fluorescence under ultraviolet light. The composition of the reagents used is described by Block et al. (14). Of course, the failure to react with any of the above reagents may have been due to the presence of an insufficient quantity of the material. With this in mind, the unknown compound was chromatographed with the following compounds, none of which coincided with the radioactive material: urea, creatine, creatinine, guanidinoacetic acid, arginine, citrul- line, proline, hydroxyproline, p-alanine, asparagine, glutamine, ureidosuc- cinic acid, ureidoglutaric acid, glyceraldehyde, glyceric acid, and tartaric acid. It was found that the compound responded to the ninhydrin-am- maniacal silver nitrate spray previously described. However, it did not migrate with dihydrouracil, hydantoin, hydantoic acid, or 5-acetic acid hydantoin. It was stable to hydrolysis in 1 N HCl (1 hour, loo”), and was absorbed on Dowex I and eluted with dilute HCl. Further attempts to identify the compound are being made.

Unknown Compound 2, found in the 90 per cent methanol-insoluble fraction, and which moved only slightly in either solvent, appeared to consist of several components whose RF values were very similar. It may represent a mixture of phosphate esters. The amount present increased gradually during embryonic development but was always a relatively minor component; no attempt was made to identify it further.

Unknown Compound 3 was present only in faint traces and was found in the ether-soluble fraction.

Unknown Compound 4 was found in the ether-soluble fraction and reacted as an acid to bromocresol purple. Chromatography with glutaric, methylsuccinic, and itaconic acids indicated non-identity. The position on the chromatogram and its stability to autoclaving for 30 minutes indi- cated that it was not any member of the tricarboxylic acid cycle.

Radioactive Components of Nucleic Acid Fraction-At all stages, between 85 and 90 per cent of the total activity of the nucleic acid fraction could be recovered in the hydrolysate. Following chromatography, only six radioactive components could be found. They were adenine, guanine, uracil, cytosine, thymine, and an unknown compound. In the aliquots used, thymine could be detected (both by ultraviolet absorption and radioactivity) only in the gastrula and neurula stages. The unknown radioactive compound (representing 10 to 15 per cent of the total activity) moved near guanine and could be detected in the blastula stage but not in the unfertilized egg or neurula. A trace was found to be present in the gastrula stage. It has no detectable ultraviolet absorption.

Typical radioautographs of the hydrolyzed nucleic acid fraction are

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346 METABOLISM OF Cl402 IN AMPHIBIA

shown in Fig. 2. Examination of the relative darkening of the film pro- duced by the radioactive compounds present shows that marked changes occurred in the distribution of the activity as embryonic development

FIG. 2. Radioautographs of the chromatograms produced from the hydrolyzed nucleic acid fractions of the frog embryo. Plate 1 is from the unfertilized egg, Plate 2 from the blastula, Plate 3 from the gastrula, and Plate 4 from the neurula. The chromatograms were developed in a manner identical to that described in Fig. 1. The following abbreviations are used: adenine, A; guanine, G, cytosine, C; uracil, U; and the unknown compound, X.

progressed. The specific activities of the purines and pyrimidines were then determined by elution of the compound with 0.005 N HCl, the amounts present being estimated spectrophotometrically and by plating and count- ing an aliquot. Adenine was measured at 260 mp, with an extinction coefficient of 13,000; guanine at 248 rnp, e = 11,000; cytosine at 275 mp, L: = 10,120; and uracil at 260 rnp, t = 8920. An HCl extract of the paper

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S. COHEN 347

was employed as a blank. Duplicate determinations of the specific activi- ties agreed within 15 per cent. The aliquots used for the determination contained between 0.05 and 0.1 pmole of purine or pyrimidine per ml. Possible contamination of the isolated compounds with other ultraviolet- absorbing material was checked at each stage by determining the ratios

TABLE III

Specijic Activities of Purines and Pyrimidines during Development

The values given are the average of two determinations from separate chromato- grams. The duplicate determinations did not differ by more than 15 per cent. The unit (counts per minute per 0.05 rmole) was chosen because approximately 0.05 rmole of the compounds was used for the analytical determinations. The relative specific activities are based in each instance upon uracil as 100.

stage of development

Unfertilized

Blastula

Gastrula

Neurula

Compound

Uracil 379 Cytosine 3 Adenine 161 Guanine 52 Uracil 875 Cytosine 147 Adenine 35 Guanine 83 Uracil 707 Cytosine 160 Adenine 171 Gu anine 115 Uracil 910 Cytosine 242 Adenine 204 Guanine 146

-

,

Experiment 1

C.p.m. per 1.05 qmle

Relative specific

activity

C.p.m. prr 1.05 /mole ,

Relative specific

activity

100 151 100 1 2 1

42 91 60 14 30 20

100 723 100 17 89 12

4 26 4 9 71 10

100 540 100 23 123 23 24 158 29 16 108 20

100 955 100 27 258 27 22 260 27 16 165 17

Experiment 2

of the extinction coefficients at 250, 260, and 280 mp. The ratios agreed satisfactorily with those reported by Hotchkiss (16).

The specific activities of the purines and pyrimidines are shown in Table III. In all stages, uracil was the most active compound present. The relative specific activities varied with the stage of development. In the unfertilized egg, cytosine showed practically no activity and adenine was about one-half and guanine one-sixth as active as uracil. In the blastula, uracil was still most active, but cytosine was relatively more active than guanine, which in turn was more active than adenine. In the gastrula and neurula, cytosine and adenine were approximately one-fourth, and guanine one-sixth, as active as the uracil.

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348 METABOLISM OF Cl402 IN AMPHIBIA

Although no great effort was made to see whether the purines and pyrimidines could be quantitatively recovered from the hydrolysate, the results from all four stages examined indicated that, although adenine was present in somewhat greater amounts than the other compounds, the ratio of adenine to guanine or pyrimidine did not exceed 1.5: 1. These results indicate that there was no extensive pool of any purine or pyrimidine pres- ent to cause the observed differences in the relative specific activities. The specific activity of the thymine was not determined because of the very small amounts present in aiiquots which could be adequately sepa- rated by chromatography.

TABLE IV

Distribution of Cl4 in Uracil during Development

The degradation procedure is given in the text. All of the carbon dioxide derived from the uracil following the enzymatic treatment was counted as B&03, and was considered to be derived from the C-2 position. All the counts were corrected for self-absorption. Sufficient counts were made to reduce the statistical error to less than 2 per cent.

Stage of development

Unfertilized eggs.

Blastula..... Gastrula

Neurula ......................

Authentic uracil-2.C4. .........

Experiment No.

Uracil BaCOa Cl& in C-2 position

c.pm. G.*.WS. *t-r cent

472 382 81 701 595 85 896 717 80 7C8 637 90 905 742 82

1780 1530 86 1540 1202 78 1106 995 90 1106 1028 93

Degradation of Uracil-The uracil was eluted with water from the chromatograms and the total number of counts present was determined. An aliquot of the solution was then degraded by the procedure described. The results are shown in Table IV. Radioactive uracil labeled in the C-2 position3 with Cl4 was degraded as a control. It can be seen that, with 0.1 pmole quantities of the C-2-labeled uracil, over 90 per cent of the activity was recovered in the CO2 liberated. The results indicate that, at all the embryonic stages studied, over 78 per cent of the Cl4 was present in the C-2 position of uracil. Considering that the recovery from the control experiment was about 91 per cent, the true percentage in the C-2 position would be even greater. No significant difference could be ob- served in the distribution of the label in the uracil at the various stages.

3The writer is indebted to Dr. Leonard Bennett for the sample of uracil-2-W.

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S. COHEN 349

Radioactive Components of Lipide and Protein Fractions-The activity of the lipide fraction was very low at all stages examined and no attempt was made to fractionate the material. The most active protein fraction (from the neurula) was hydrolyzed with 6 N HCl by autoclaving (126”) for 15 hours. The HCl was removed by repeated evaporation in a vacuum desiccator over KOH. Even with the most active fraction, at least 1 month was necessary to detect any radioactive compounds in aliquots suitable for chromatography. Active aspartic and glutamic acids could be identified. Traces of other active material were present, but their activity was extremely low and no further attempt was made to identify them. Their identification would probably be greatly facilitated by a preliminary fractionation of the protein in order to eliminate the yolk granules.

DISCUSSION

The frog egg is characterized by an almost complete self-sufficiency with respect to metabolic interactions with its environment. Provided with oxygen and an aqueous medium, the egg will develop into a young tadpole. Development during this %losed system” period thus proceeds with a rearrangement of the stored material of almost purely maternal origin.

It is perhaps not surprising, therefore, that the previous attempts to label frog embryos by incubating the intact eggs in a medium containing radioactive phosphate (I), glycine (2), or methionine (3) have not been very successful. The present experiments have taken advantage of the permeability of the frog egg to carbon dioxide. It is not known whether there are any differences in the permeability to carbon dioxide at the stages examined, but such differences, if they exist, should not affect the relative distributions of the isotope.

In many ways the qualitative distribution of the isotopic CO2 in the frog embryo is similar to what one would expect from the known reactions involving carbon dioxide fixation in mammalian tissue (17). The fixation of COZ into malic, oxalacetic, and oxalosuccinic acids with the subsequent operation of the tricarboxylic acid cycle would explain the detection of the isotope in the succinic, fumaric, malic, and citric acids. The presence of active aspartic and glutamic acids would be expected if the enzyme systems required for the transamination of oxalacetic and cw-ketoglutaric acids were present. The fixation of CO2 into the purines and pyrimidines of a variety of organisms has been described (18) but the enzymatic mechanisms re- main obscure. Ureidosuccinic acid has been postulated as an intermediate in pyrimidine synthesis (19) and Lieberman and Kornberg (20) have demonstrated the production of ureidosuccinic acid from erotic acid in bacterial extracts. To the author’s knowledge, ureidosuccinate has not previously been demonstrated in tissue.

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350 METABOLISM OF Cl402 IN AMPHIBIA

No radioactive arginine has been detected in our experiments. The presence of active arginine would be expected if the Krebs ornithine cycle were in operation. Munro (21) has shown that ammonia excretion, pre- dominant in the tadpole, gives place during metamorphosis to a urea excretion, while at the same time there is a 20-fold increase in the liver arginase. Before these data can be fully accepted as indicating the absence of the Krebs ornithine cycle in the developing embryo, the absence of radioactive arginine in the protein fraction should also be demonstrated. No radioactive urea or creatine could be detected at any of the stages examined. These presumably would be present in the adult animal.

The results of the examination of the relative distribution of the isotope in the cell-soluble, lipide, nucleic acid, and protein fractions of the embryo during development indicate that, even in the unfertilized egg, enzyme systems are present which are able to fix carbon dioxide. However, while the major portion of the activity in the unfertilized egg remains in the soluble fraction of the cell, the relative rates at which the active carbon of this “pool” is converted to protein and nucleic acid carbon are increased as development proceeds. A somewhat similar conclusion was reached by Kutsky (1) in her study of the distribution of P32 during amphibian develop- ment. She states that, at the onset of gastrulation, there is a marked shift of the P32 from the acid-soluble fractions into the nucleic acid and other acid-insoluble fractions.

After this manuscript had been completed, a study similar to the present investigation was reported by Flickinger (22). The incorporation of Cl402 into the protein, lipide, deoxyribonucleic acid, ribonucleic acid, and trichloroacetic acid-soluble fractions of the developing frog embryo (Rana temporaria) was determined from the two-cell stage to the tail bud larvae. The unfertilized egg was not examined. The results obtained were very similar to the present researches. The specific activity (counts per minute per mg. of carbon) of the acid-soluble fraction decreased during embryonic development, whereas the activity in the protein and nucleic acids in- creased. The lipide fraction remained low in activity in all stages. The composition of the cell fractions isolated was not examined.

The significance of the accumulation of Compound 1 and its relationship to the decrease in activity of aspartic acid and the lesser accumulation of ureidosuccinate during the blastula and gastrula stages must await identi- fication of the unknown compound. The failure to find appreciable amounts of active citric acid in the unfertilized egg may be a reflection of a very low rate of operation of the tricarboxylic acid cycle as compared to later stages.

The distribution of the Cl4 in the nucleic acid fraction during develop- ment changes both quantitatively and qualitatively. In the unfertilized

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S. COHEN 351

egg, where presumably no increase in the net amount of nucleic acid is occurring, from 21 to 25 per cent of the fixed Cl4 is present in this fraction. The isotope has been detected in the adenine, guanine, and uracil of the fraction, but very little, if any, is present in the cytosine. While the relative amount of Cl4 fixed in the nucleic acid fraction increases gradually as development progresses to the neurula stage, considerable differences are noted in the relative specific activities of the purines and pyrimidines present. Uracil, however, always has the highest specific activity.

Steinert (23) noted the presence of free hypoxanthine and guanine in the mature oijcytes of Rana fusca and in T&on alpestris. These began to disappear after early gastrulation and were suggested to be precursors of adenine and guanine in the nucleic acids. This may explain the relatively low incorporation of carbon dioxide into the purines as compared to uracil. In our experiments, no radioactive free guanine or hypoxanthine has been detected at any stage. The significance of the extremely low activity of the cytosine in the unfertilized egg is not understood. The high specific activity of the uracil at all stages may possibly be due to an exchange reaction occurring at carbon 2 without involving resynthesis of the entire uracil molecule.

It should be emphasized that the nucleic acids were not isolated as such, and the possibility remains that some of the results obtained may be due to the presence of small amounts of highly active nucleotides in addition to the ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in the hot trichloroacetic acid extract. It should also be remembered that the results were obtained from an analysis of the whole organism; localization in the embryo of some of the observed changes would be of great interest. The stages of development studied were chosen arbitrarily. Intermediate stages may possibly show still other characteristics.

Our results indicate that at least 78 per cent of the activity in the uracil is located in the C-2 position. Heinrich and Wilson (24) reported that, following the injection of radioactive carbonate into the rat, almost all the activity of the isolated uracil was present in the C-2 position, and the rate of incorporation of the isotope into the C-2 of uracil and the C-6 position of guanine was similar. On the other hand, Lagerkvist (25) found that carbonate enters the uracil of the regenerating liver of the rat to the same degree in both the C-2 and C-4 positions. However, the adequacy of the method used by Lagerkvist for the degradation of uracil has been ques- tioned (26).

The gradual increase in the specific activity of the protein of the devel- oping embryo is perhaps what one would expect from the cytological evidence. The oiicyte is laden with large amounts of presumably inert yolk granules whose substance is gradually incorporated into the “active

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352 METABOLISM OF C1402 IN AMPHIBIA

protoplasm” of the embryo. The increased specific activity of the protein would result if the amino acids of the yolk protein pass through the soluble amino acid “pool” before being converted into the protein of the embryo. However, it is also possible that the conversion is more direct and t)he increased specific activity of the protein is due to the greater exchange of the completed protein with the amino acid “pool.” The presence of a variety of free amino acids in the frog egg and embryo has been described by Eakin, Berg, and Kutsky (27) and by Holtfreter, Koszalka, and Miller

cm. These data serve to delineate some of the metabolic changes which

accompany differentiation in the frog embryo. With respect to the general applicability of the results to other embryos, Hultin (29) has recently reported his results on the incorporation of C14-labeled carbonate into sea-urchin eggs. Carbon dioxide was fixed at all stages from the unfertilized egg through the gastrula, with an increase in the fixation mainly during blastula formation. The results obtained were similar to the present results in several respects. The specific activity of the pro- tein carboxyl carbon gradually increased during development. Only traces of activity could be found in the lipide fractioa. Activity was found in the adenine and guanine of the RNA. On the other hand, activ- ity was also found in the free hypoxanthine of the cell, and there was a peak of activity in the soluble fraction during the blastula stage. How- ever, the two experiments are not directly comparable because, in Hultin’s researches, a trichloroacetic acid extraction was employed to obtain the soluble material of the cell, whereas aqueous methanol was employed in the present researches.

The problem of the correlation of these chemical changes with the visible morphological changes remains. It is possible that a more intimate study of the location of these metabolic changes may help in achieving further progress.

SUMMARY

1. The metabolism of carbon dioxide during early embryonic develop- ment was studied. Eggs and embryos of the frog, Rana pip&s, were exposed to U402, and the distribution of the activity was determined. Four stages of development were examined: the unfertilized egg, the blastula, the gastrula, and the neurula.

2. In the unfertilized egg, from 71 to 75 per cent of the activity was present in the soluble fraction, 21 to 25 per cent in the nucleic acid frac- tion, 3 to 4 per cent in the protein, and less than 0.5 per cent in the lipide Eraction. As development progressed from the unfertilized egg to the neurula, there were a marked lowering of the relative isotope content of

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S. COHEN 353

the soluble fraction and an increase in the relative activity of the nucleic acid and protein fractions.

3. The following radioactive compounds have been identified in the soluble fraction: aspartic, glutamic, malic, succinic, fumaric, citric, and ureidosuccinic acids. The accumulation of an unknown radioactive compound during the blastula and gastrula stages has been noted.

4. The specific activities of the adenine, guanine, cytosine, and uracil of the nucleic acid fraction have been determined. Uracil is most active at all stages. Cytosine shows practically no activity in the unfertilized egg. The relative specific activities of these purines and pyrimidines differed at each stage of development.

5. An enzymatic procedure for the degradation of 0.1 PM quantities of uracil has been devised. At least 78 per cent of the activity of the uracil was found to be located in the C-2 position.

The writer is indebted to Dr. Martin Kamen for helpful advice and criticism.

BIBLIOGRAPHY

1. Kutsky, P. B., J. Exp. Zool., 116, 429 (1950). 2. Friedberg, F., and Eakin, R. M., J. Exp. Zool., 110, 33 (1949). 3. Eakin, R. M., Kutsky, P. B., and Berg, W. E., Proc. Sot. Exp. Biol. and Med.,

‘78, 502 (1951). 4. Hamburger, V., A manual of experimental embryology, Chicago (1942). 5. Shumway, W., Anat. Rec., 78, 139 (1940). 6. Schneider, W. C., J. Biol. Chem., 161, 293 (1945). 7. Hanes, C. S., and Isherwood, F. A., Nature, 164, 1107 (1949). 8. Marshak, A., and Vogel, H. J., J. Biol. Chem., 189, 597 (1951). 9. Hayaishi, O., and Kornberg, A., J. Biol. Chem., 197, 717 (1952).

10. Needham, J., Chemical embryology, Cambridge (1931). 11. Gregg, J. R., and Ballentine, R., J. Exp. Zool., 103, 143 (1946). 12. Barth, L. G., J. Exp. Zool., 103, 463 (1946). 13. Hultin, T., and Wessel, G., Exp. Cell Res., 3, 613 (1952). 14. Block, R. J., LeStrange, R., and Sweig, G., Paper chromatography, New York

(1952). 15. Nyc, J. F., and Mitchell, H. K., J. Am. Chem. Sot., 69, 1382 (1947). 16. Hotchkiss, R. D., J. BioZ. Chem., 176, 315 (1948). 17. Krebs, H. A., Symposia Sot. Exp. Biol., 5, 1 (1951). 18. Christman, A. A., Physiol. Rev., 32, 303 (1952). 19. Wright, L. D., Miller, C. S., Skeggs, H. R., Huff, J. W., Weed, L. L., and Wilson,

D. W., J. Am. Chem. Sot., 73, 1898 (1951). 20. Lieberman, I., and Kornberg, A., Federation Proc., 12, 239 (1953). 21. Munro, A. F., Biochem. J., 33, 1957 (1939). 22. Flickinger, R. A., Exp. Cell Res., 6, 172 (1954). 23. Steinert, M., Bull. Sot. chim. biol., 33, 549 (1951). 24. Heinrich, M. R., and Wilson, D. W., J. BioZ. Chem., 186, 447 (1950).

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25. Lagerkvist, U., Acta &em. hand., 4, 1151 (1950). 26. Fairley, J. L., Daus, L. L., and Krueckel, B., J. Am. Chem. Sot., 75,3842 (1953). 27. Eakin, R. M., Berg, W. E., and Kutsky, I?. B., Proc. Sot. Exp. Biol. and Med.,

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Stanley CohenAMPHIBIAN DEVELOPMENT

DURING2O14THE METABOLISM OF C

1954, 211:337-354.J. Biol. Chem. 

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