THE ENERGY-SOURCES IN ONTOGENESIS.

I . — T H E U R E A CONTENT OF THE DEVELOPING AVIAN EGG.

By JOSEPH NEEDHAM, M.A., PH.D.,

Fellow of Gonville and Caius College, Cambridge.

{From the Biochemical Laboratory, Cambridge!)

(Received November ist, 1935.)

T H E class of compounds used by the developing embryo for theextraction of energy from its environment raises one of the mostimportant questions which arises in the biochemical analysis ofembryological phenomena. Does it behave exactly like theadult animal from the very beginning, or does it pass throughphases each of which are, in this respect, different? To beable to compile a chart showing the compounds which thedeveloping embryo utilises as energy-sources throughout itsontogenesis, to know the times and periods in which one super-sedes another, would be to possess clues of enormous importancefor the unravelling of every event in embryonic metabolism.

At the present time it is generally believed that fat is theenergy-source specially associated with embryonic growth. Thisview, which has arisen almost entirely from work on the hen'segg, is due to the researches of Pott (34), Liebermann(25), andTangl and von Mituch(44). Estimations of the fat in the embryoand that in the rest of the egg showed that some had been lostin transit, actually from 2.1 to 2.76 gms. per egg on an average.Calculation demonstrated that this figure corresponded satis-factorily with the carbon dioxide evolved throughout incubationand the heat produced during the same time. Needless to saythe fit of the figures was not very exact but was thought to besufficient to show that fat was the only source of energy. Forexample, Libermann (25) had shown that the fat of the egg con-tained 71.67 per cent, of carbon, and the amount used per eggTangl and von Mituch(44) found to be (on an average) 2.11 gms.According to Hasselbalch(io.), the total amount of COS producedduring incubation amounted to 5.939 gms., i.e. 1.62 gms. of

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Joseph Needhamcarbon. From this the calculated amount of fat used was2.26 gms., which agreed moderately well with Liebermann'sfigure but rather less so with one which they themselves found,viz., 2.76 gms. Another line of investigation which gave greatsupport to this view was the work of Bohr and Hasselbalch (4),who measured carefully the respiratory quotient of the hen'segg during development. From the seventh day onwards theirfigure was very constant at about 0.73, so that unless somethingvery unusual was going on, fat must be considered as the onlyenergy-source.

It might always have been doubted, however, that fat wasthe only important source of energy: there were hints to thecontrary in the literature. William Harvey (18) had said, "andtherefore the yolke seems to be a remoter and more deferredentertainment than the white, for all the white is quite andclean spent before any notable invasion is made upon theyolke." Another important observation was that of PreVostand Le Royer (35) in 1825, who obtained from the allantoic fluidof a seventeen-day old chick a nitrogenous substance whichgave an insoluble nitrate and resembled urea in all particulars.There had clearly been some catabolism of proteins. And sinceat about the same time Jacobson (21), Sacc(37), and Stas(4i)found uric acid in considerable amounts in the allantoic andamniotic fluids of developing chicks during the third week ofincubation, there was no doubt about it. Moreover, Wetzel(52)from a study of the chemical composition of many different typesof eggs before and after development came to the conclusionthat all three kinds of foodstuff had been burnt.

In a previous paper (30) I pointed out that whether or not fatwas the predominating energy-source it could certainly not beconsidered to be the only one, for many arguments of greatforce pointed to bodies of carbohydrate nature as being theenergy-sources of the initial stages. These arguments can besummarised as follows :—

1. In the first eight days of incubation of the hen's egg there is a strikingfall in the amount of free glucose. The curves, which will be found in thepapers of Sato (38), Idzumi(ao), Bywaters(5), and Tomita(46), all show arapid fall from about 0.4.gm. per cent to 0.1 or less.

2. Simultaneously with this disappearance of free glucose, the lactic acid inthe egg, which has previously been low, reaches a peak and immediately after-

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The Energy-Sources in Ontogenesiswards descends to its previous level. The curves given in Tomita's paper (5)show that from an initial level of 0.02 mgm. per cent it attains on the fifth daya maximum of 0.13 mgm. per cent and regains its original value about thefourteenth day. That it was intimately connected with glucose he proved byinjecting glucose and observing an increase of lactic acid.

3. If the figures of Bohr and Hasselbalch (4) are closely examined, it will beseen that although the respiratory quotient during the last two weeks ofincubation is certainly 0.73 on an average, this is not the case with the earlierstages. On the contrary, figures as high as 0.91 and 0.81 were obtained,although in small number. Nothing could fall better into line with the resultsof Tomita; if anything these early figures are low, for two reasons. Firstly,Hasselbalch (19) showed that the egg, especially the yolk, gave out small amountsof oxygen during the first twelve hours of development and, secondly, the eggprobably possesses an alkali reserve which would tend to absorb the first smallamounts of expired CO2. These high figures in the early stages did not escapethe notice of Grafe(is), who in his review of the subject in 1910 thought thatthe high respiratory quotient might be associated in some way with the lavingdown of morphological structure.

4. The disappearance of glycogen in very early stages of development hasbeen investigated by Lewis (24) and Konopacki(22). The former grew tissue-cultures of cells from very young chick embryos and could never find glycogenpresent in them except if they were taken before the first fifty hours of develop-ment were over. Konopacki, working on the frog, obtained exactly similarresults. He found that after fertilisation and the formation of the perivitellusthe glycogen greatly diminished in quantity and remained very low until theneurala stage was reached, after which the glycogen rose again. Similar resultshave recently been reported by Vastarini-Cresi(47). All these workers made useof microchemical methods.

5. Evidence pointing in the same direction is seen in the work of Warburg,Posener, and Negelein (49). They occupied themselves in examining theglycolytic behaviour of neoplasmic arid embryonic tissues, and found a verymarked preferential consumption of carbohydrate on the part of 3 to 5 day chicks.The production of ammonia when the tissue was suspended in bicarbonateRinger in vitro was greater than that of any adult tissue save brain or retina, andcorresponded to the similarly high position of embryos of this age as regardsglycolytic power. But if sugar was provided for it, it would immediately ceaseto give off ammonia and would utilise carbohydrate. This fact, and the factthat the power of glycolysis is very high in early development and much lowerin the adult condition both confirm strongly what has been said before.

6. Perhaps it is no coincidence that the respiratory quotient which is foundin the early cleavage stages of small marine eggs such as those of the sea-urchin isin the near neighbourhood of unity. Warburg (48) found a respiratory quotientof 0.9 for the first twenty-four hours of the development of Arbacia pustulosaeggs, and Shearer (40) working on those of Echinus microtuberculatus obtained afigure of 0.95. A higher figure still was found by Faur6-Fremiet (9), whoseresults with the eggs of Sabellaria alveolata work out at an R.Q. of 1.0. Asindices of the nature of the combustions going on, these figures ought to be

191

Joseph Needhamaccepted with the utmost caution, for there may be curious gas effects atfertilisation, e.g. the "neutral gas "of Bialascewicz and Bledowski (2). But it isnatural to suppose that a stage which lasts five days in the chick may last onlya few hours in lower animals which develop faster, so that the time to look forutilisation of carbohydrate would be from fertilisation to the neurula stage.

7. What would appear to be data antagonistic to the view now put forwardare found in the monograph of Le Breton and Schaeffer (23). Calculating fromthe results of Bohr and Hasselbalch (4) on the chick they found that the numberof gram calories produced per gram of embryo wet weight per hour was muchhigher in the initial stages of incubation than towards the end. They associatedthis fact with the curves which they obtained for the chemical nucleoplasmic

.. . purine nitrogen x 100 .ratio, i.e. t o t a l m t r o g en - purine nltr0Sen> b u t t h ey d l d n o t d r a w the conclusionthat it looked as if fat was being used as the energy-source in the beginning. Arough calculation shows, however, that though this is so at first sight, it is notreally the case. From the figures of Eaves (8) and Idzumi (20), it is found thatabout 0.3 gm. of fat are combusted by the chick on the fourteenth day. Inthis calculation a small correction must be made for the amount of fat stored inthe embryonic tissues; this is done by means of the accurate embryonic fat figureskindly lent me by Dr H. A. Murray. The still smaller corrections necessary toallow for the fat set free from the lipoids at this time (Plimmer and Scott (33))and the fat simultaneously removed as esters of cholesterol (Mueller (2 7)),practically cancel each other out. In the .fourteenth day, therefore, the fat com-busted amounts to approximately 0.038 gm. per gram of embryo wet weight. Onthe other hand the figures of Sat6(38), Bywaters(s), and Tomita(46) show thatin the fifth day about 0.04 gm. of free glucose is combusted, which works out ato. 18 gm. per gram of embryo wet weight, or five times as much as the amount offat similarly used on the fourteenth day.* Le Breton and Schaeffer's curve istherefore explained by the fact that in the early stages of incubation relativelymore carbohydrate is used per gram of embryo than is fat per gram of embryolater on. If these figures were related to dry weight instead of to wet weight thedifference would be more striking, for the older the embryo becomes the drier itgets. This argument is almost the same thing as saying in Tangl's (43) termin-ology that the relative and specific " Entwicklungsarbeit" is higher in the earlierstages than it is later on. It is also interesting to note that in Le Breton andSchaeffer's curve the gram calories per gram of embryo wet weight per hourdescend very rapidly to the end of what from the above evidence we might callthe carbohydrate period. On the fourth day the figure is 46.6 and on the seventhday only 9.6, after which the fall is not nearly so rapid and on the eleventh daythe figure is still four. The inflection in the curve comes when the glucose hasdisappeared and not long after the peak of lactic acid. The descending curveof Le Breton and Schaeffer is reflected again in that of Shearer (40), who studiedthe oxygen-consumption of embryonic tissue in vitro. The metabolic rate,i.e. the c.c oxygen consumed per gram wet weight per hour calculated fromhis figures, falls almost exactly on the curve of Le Breton and Schaeffer.

• Moreover this figure is low, if anything, for the glycogen disappearing at thesame time as the glucose has not been taken into consideration.

192

The Energy-Sources in OntogenesisThese arguments seem to show fairly clearly that in the hen's

egg and in other eggs there is a preliminary period when carbo-hydrate is used as the principal source of energy. There are,however, other data in the literature which are not explained uponthis hypothesis, such as the curious results relative to the calorificquotient obtained by Meyerhof(26) and Shearer (40), and thework of Bialascewicz and Mincoffna (3) on the frog. For a longtime, too, all the data have pointed to an exclusive combustion ofcarbohydrate by mammalian and viviparous embryos. Thesemay have evolved special metabolic habits to meet their specialconditions, and have perhaps prolonged their carbohydrate periodto cover the whole of their development. In other cases, theperiod of carbohydrate utilisation may have been shortenedto a few hours; if this were the case certain contradictoryfacts at present in the literature would be explained. It waspointed out above that the fit of the figures in Tangl and vonMituch's calculation was not very exact; so this cannot beadduced as an argument against the existence of a "carbo-hydrate period," since during its predominance the embryo isvery small and so also is the total turnover of matter andenergy. But there are further arguments, which not onlyconfirm those which have been given above, but which point tothe existence of a period of protein combustion, midway betweenthe utilisation of carbohydrate and the utilisation of fat.

r. Simon Gage and-Susanna Gage(13) fed laying hens on Sudan III andfound that although red eggs were laid, the embryos showed no trace even of apink colour until the tenth day of incubation. After that time they becamerapidly more coloured until at hatching they were quite red. One wouldsuppose that in order to be combusted, fat would have to be absorbed into theembryo and would take the dye with it, as indeed did happen after the tenthday. Confirmation of this is found in the figures of Dr H. A. Murray, referredto above, for fat storage in the chick. At the eleventh day it begins to rise anddraws away from its previous leveL Both these facts point to an awakening offat metabolism at the mid-point of incubation,

2. The work of Riddle (36) on the yolk and the yolk-sac is interesting in thisconnection. From the fifteenth to the twentieth day the yolk is the only supplyof the chick, and his chemical study of it during this period shows that there isa preferential absorption of fat. The neutral fat decreases markedly in theyolk and the protein substances increase.

3. Bialascewicz and Mincoffna (3) working on the frog's egg found that up tohatching there had been practically no loss of fat, but that a loss of proteincould be recognised. They did not look for any preliminary period of

193

Joseph Needhamcarbohydrate consumption, but concluded that protein during the early periodhad undoubtedly served as a source of energy.

4. The earliest statement that protein in the hen's egg must be a source ofenergy is due to Schneroffna(39). She found a constant relationship betweenthe nitrogen in the embryo and the nitrogen in the allantoic fluid, a ratio whichalways worked out to about 17. Every 17 gms. of nitrogen absorbed by theembryo corresponded exactly to 1 gm. excreted. She concluded that thisindicated protein as a source of energy, and gave figures to show that therewas a peak of total nitrogen in the embryo at the ninth day. She interpretedthis as showing a peak of protein absorption at that point—certainly in viewof what we have already said, the point at which it might be expected tocome. But there is no a priori reason for wishing to associate the point ofgreatest protein absorption with the period of greatest protein combustion{cf. Bywaters(5)).

5. Faure-Fremiet and Dragoiu (10) made an extended study of the frog's egg.They agree with Bialascewicr and Mincoffna in finding a loss of protein beforehatching but they also observe a loss of glycogen and of fat, indicating that allthree substances have acted as sources of energy. The same conclusion wasarrived at by Tangl and Farkas (45) for the egg of the silkworm, Bombyx tnori.None of these workers observed any stages in this. During most of thedevelopment Faure'-Fremiet and Dragoiu obtained an R.Q. of 0.6, whichagrees well with the 0.65 and 0.7 of Parnas and Krasinska (32) andof Bialascewicz and Bledowski respectively. This state of affairs is like thatseen in the hen's egg, where for slightly over two-thirds of the total incubation-period, the R.Q. indicates combustion of fat. Moreover, Dakin and Dakin (7)found a utilisation of proteins during the development of the eggs of the plaice,and Greene (16) observed the same thing in the king-salmon.

6. If urea and uric acid are indicators of protein metabolism, so also is thephenomenon of specific dynamic action. Evidence that there is a period indevelopment when this phenomenon appears is contained in the recent workof Gayda (53). Using a differential calorimeter, he measured the heat given outby developing toad embryos throughout their development. When he calculatedthe heat given out during each period in which the weight was doubled, heobtained a curve with a peak in the centre, to which the values rose andfrom which they descended. Thus development was more economical at thebeginning and end of development than at the middle. When this is comparedwith the figures of chemical analyses (9, 28, and 2), it is seen that there is apredominant combustion of protein during the middle part of development,proper allowance being made for the slower development of the toad ascompared with the frog. I have made similar calculations for the hen, using thefigures of Bohr apd Hasselbalch (4), and Dr Harry Murray. Exactly the samepeaked curve appears, and shows that there is a point of least economicaldevelopment about the ninth day; this may well be due to the specific dynamicaction of protein combusted at that period.

There is thus a great deal of evidence in the literaturewhich points to a succession of stages, each with a definite

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The Energy-Sources in Ontogenesisenergy source. The only facts which diametrically oppose theview that in the earliest stages carbohydrate is utilised are thosebrought forward by Meyerhof(26), in the paper referred toabove. He found that the calorific quotient of Arbacia pusto-losa eggs immediately after fertilisation was 2.6, and concludedthat fat was being utilised, although the classical figures ofRubner are 3.2 for protein, 3.3 for fat, and 3.5 for carbo-hydrate. Until a closer approximation to one of these isreached, it would surely be better to suspend judgment. WithAplysia limacina the figure was 3.0 which was little better.Meyerhof supported his opinion that fat was being combustedin the earliest stages by showing that the intensity of stainingwith sudan III diminished as development proceeded, thoughsignificantly enough it made a rapid fall after the pluteus stage.He looked for a carbohydrate in echinoderm eggs but could notfind any with the usual tests—by no means proof that nonewas there. It does not seem that these data are sufficient tocounterbalance the facts which have already been mentioned.

Probably other substances besides protein, fat, and carbo-hydrates may be utilised to supply energy in some forms oflife. For example, the recent discovery by Heilbronn ofLiverpool, of great amounts of spinacene, a cholesterol-likesubstance, in teleostean eggs, may lead to the solution of theproblem of the energy-source of these embryos. What theycombust has so far been quite unknown (see re/. 30, pp. 20and 21).

Grafe in 1910(15) thought that there might be some con-nection between the period of carbohydrate utilisation in thechick's development and the fact that at that time all theorgans were being formed, so that profound morphogeneticchanges were going on. In the paper already referred to (30),I suggested that the carbohydrate period at the beginning andthe fat period at the end might be associated with preponder-ance of change of shape, internal and external, on the onehand, and change of size on the other hand. But perhapsthe time has not yet arrived for correlations of this kind. Inthe first place, "augmentation" and "framing," "growth" and" differentiation " are not nearly such simple processes as theyhave been considered to be in the past, for as Murray (29) has

19S

Joseph Needhamshown, morphological and chemical differentiation are two quitedistinct things, as are also morphological and chemical growth.Morphological differentiation and morphological growth changemost rapidly at the beginning of embryonic life, while chemicaldifferentiation as expressed by metabolic rate and changes inconcentration of chemical substances, and chemical growth asexpressed by the increase of dry solids relatively to water,change most rapidly in the later stages {see also Ref. 30, p. 50).Secondly, the data are at present only fragmentary and untilmany more are collected, it must remain unwise to correlatefacts too hastily. On the other hand, it would be veryinteresting if any connection appeared between a succession ofenergy-sources in ontogenesis and the numerous observations ofsusceptible stages in development, such as those of Stockart (42)and of Parnas and Krasinaka (32). This work has brought outwith exceptional clearness the fact that development may bediscontinuous and in all cases passes through critical stageswhen disastrous effects will follow an interference innocuousat other times. Such a critical stage is the beginning ofgastrulation. Stockard says : " The present extremely crudestate of our knowledge of the chemistry of development willpermit of no satisfactory statement of the principles underlyingdifferences in developmental rate." Perhaps the speculationmay be permitted that critical stages in development may turnout to be associated with changes from one type of substanceto another type as a source of energy. An intermediate linkin the chain of events would be the rapid growth-rate of oneor more organs, leading to a teratological result if developmentwas at that moment interfered with.

But what are needed first of all are investigations to decidewhether the conception of a succession of energy-sources iswell-founded or not If indeed it occurs in the chick, will itbe found to occur also in all types of embryo ? How far willit be found to be affected by the adaptations of particularspecies to the peculiar necessities of their environment ? Theseare the problems which must first be answered.

But assuming that the conception of a succession of energy-sources should prove to be well-founded, its exact interpretationwould still remain uncertain. The ontogenetic procession

196

The Energy-Sources in Ontogenesiscould be either "ovogenic" or "embryogenic." On theformer view the energy-sources would succeed one anothersimply because the dynamic equilibria and the relative concentra-tions of substances in the yolk and white necessitated it. Theembryo would play a passive part, combusting protein andfat only since it could not get carbohydrate. On the latterview the succession of energy-sources would be intimatelyconnected with the changing potentialities of the growingembryo. Energy must be the same from whatever source itcomes, but the embryo—on this view playing an active part—would combust such and such substances at such and suchperiods of its development because it would not have at thosetimes the capacity for combusting others. The molecularorientations on its intracellular surfaces would differ at differentstages of its development, perhaps in relation to its differentia-tions and growths, or at diverse times its enzyme systems wouldvary profoundly in activity.

At present there is not enough evidence to allow us to makea choice between these views. The ovogenic hypothesis wouldcommit us to the belief that if sufficient carbohydrate werepresent during the protein and fat combustion periods theutilisation of these latter by the embryo would greatly diminishor disappear. On the embryogenic hypothesis we should haveto believe that however much carbohydrate were present duringthe protein period the embryo would continue to combustprotein, for a close relation would exist between its source ofenergy and its stage of development. In favour of the ovogenichypothesis might be cited the case of the viviparous embryo,which is believed to combust carbohydrate throughout itsdevelopment (3). But dangers beset any direct comparisonbetween embryos in ovo and in utero. Mammalian embryoshave an unlimited supply of nutriment, owing to their continuousperfusion system (1, 18), avian embryos do not; so that in onecase the proportion of embryo to nutriment does not alter and inthe other case it does. Mammalian embryos can have all theircombustible material supplied to them in solution ; if the avianembryo lived in the same style it would need an egg vastlylarger than its present size to contain its physiological sugarsolution. The fat of the avian egg is tabloid food. Moreover,

197

Joseph Needhamthe placenta, as the reviews of Murlin(28) and Zuntz(5i) show,exercises a far-reaching selective influence on the food of theembryo, such as is not known to exist in the avian oramphibian egg.

The active autohegemonic powers of growth which theembryo has been shown to possess by the experimental embryol-ogists such as Hertwig, Roux, and Driesch might seem tofavour the embryogenic hypothesis. In its support could alsobe adduced the fact that during the protein period in the avianegg, there is plenty of carbohydrate being absorbed in thebound form of ovomucoid. But accurate estimations of thecarbohydrate content of the embryo must be done before thispoint can be elucidated. Perhaps light will be thrown on itby the researches of Hanan on the embryonic blood-sugar,a preliminary report of which has already appeared (17).Attempts will also be made in this laboratory to study theeffects of injecting glucose. If teratological results can beavoided (and it is believed that they can (31)), the demonstrationof a protein or fat-sparing action would help to decide betweenthe two hypotheses.

Although the developing chick has been so much investi-gated, there remains a very wide field for observations uponit. Since we already have many data obtained on the chickwhich bear directly upon the question at issue, it was consideredbest to concentrate here for the time being. With this end inview, experiments have been carried out on the urea contentof the egg during incubation, and these are reported in thispaper. Determinations of the uric acid content of the egg areat present in progress and a subsequent paper in this series willbe devoted to them. Eventually a study of the comparativeglycolytic power of embryonic tissues at varying stages ofincubation will be undertaken.

It is in the highest degree remarkable that so few researcheshave been carried out on the urea and uric acid in the hen'segg. Probably this has been due to the absence of accurateand simple estimation methods, especially in view of thepractical difficulties attending work on eggs. Gori(i4) madequantitative estimations of the urea in the eggs of Torpedoocellata and of various fishes, but he did not follow the changes

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The Energy-Sources in Ontogenesisthat occurred throughout incubation. The only other studyis that of Fridericia (12) who estimated the uric acid in theembryo and its spaces from the eleventh day onwards, butneglected the urea as being a priori unimportant, since thenitrogen excretion of the adult hen is 90 to 95 per cent inthe form of uric acid. He did, however, make a few estima-tions of the urea present on the seventeenth day of incubation,using Schondorffs laborious method, and found the resultsto be low and variable. It was clearly necessary to go intothe matter afresh, using a more modern method and tryingto get a complete curve for the whole of incubation. In any•case the fact that his estimations began only at the eleventhday renders them of little use for our purpose, since the mostinteresting changes might be expected to occur before thatpoint.

Experimental results.

The eggs used in these experiments were those of WhiteLeghorn hens placed in the incubator on the same day thatthey were laid. For analysis the shell was broken open atdifferent places at different periods of incubation. At thebeginning it is best to open it along one long side and to letthe yolk and albumen flow out into a basin, leaving the embryoand its membranes in the shell, from which they can be easilyremoved. In the earliest stages of all, however, it is best tobreak the whole egg contents into an evaporating basin andremove the embryo from the top on which it floats. Laterthere supervenes a period in which the yolk membrane isextremely fragile, so that the greatest care has to be usedin opening the egg. As the yolk flows out it must be easedwith a pair of forceps to avoid tearing the membrane againstsharp points of the broken shell. Towards the end of incuba-tion the yolk membrane again gets tougher, and as by thistime the allantois has grown completely round the interior ofthe shell, it must be cut and turned back all round theopening before the yolk is pulled out. In this way thereis a minimal loss of the allantoic and amniotic fluids.

The tared vessel containing the embryo, its membranes,and their contents, is weighed and the whole material carefully

VOL. III.—NO. 3. 199 O

Joseph Needhamand quantitatively ground up with fine washed sand. Then itis washed into a large test-tube, using plenty of wash water.This is placed in a water-bath at iooc to coagulate theprotein, which is removed by a prolonged series of decantations,or more quickly by filtering through closely woven muslin.The combined extracts are then evaporated down with gentleheat to a small bulk, perhaps 25 c.c, and in this liquid the ureais estimated by the method of Folin and W u ( n ) . Anotherpreliminary procedure has to be adopted after the fourteenthor fifteenth day, for owing to the growth of the bones, itbecomes impossible to grind up the chick satisfactorily withsand. The embryos are placed in a Bolinder mincing machine,which in a short time reduces them to a uniform pulp. Themass is then extracted twice with acetone in the proportionof 10 gms. of tissue to 30 c.c. of solvent. No further ureais extracted by a third or fourth repetition of this process.The coagulated tissue is filtered off and the acetone recoveredby distillation, so that a watery solution is left which can betreated in exactly the same way as described above. Anytraces of acetone are expelled during the evaporation to smallbulk.

In Folin's method the urea is decomposed by urease actingfor thirty minutes at 50° in presence of a few drops of phosphatebuffer mixture at / H 7.0, a saturated solution of borax added,and the ammonia driven over by rapid distillation into ice-cold0.05 N hydrochloric acid, where it is estimated colorimetricallyby nesslerisation. The blanks with this method are excellent,but for good results it is essential to have freshly acetone-precipitated soya bean. [See Van Slyke and Cullen(5o)].Each estimation must be accompanied by a duplicate withouturease in order to know the amount of free ammonia presentin the sample, and this value must be deducted from the other.

The results are all tabulated in Table I. Columns D andE are calculated from the very accurate series of figuresobtained for wet and dry weights of embryos by Dr H. A.Murray, who kindly put them at my disposal. The dailyexcretion, calculated from column C in column F shows asteady rise until the eleventh day, followed thereafter by a seriesof oscillations. The daily excretion rate naturally follows this.

2 0 0

The Energy-Sources in OntogenesisThe results are shown graphically in figs, i and 2. In

fig. 1 is seen the mgms. per cent, weight plotted against thetime and also the mgms. of urea related to each embryo. Themgms. of urea per embryo rise steadily as might be expected,but the mgms. per cent, of wet weight follows a much moreinteresting curve. It rises steadily also, until the ninth day,

TABLE I.

Day.

A.

45

6

7

89

IOI I

1 2

1314151617,a1 O

192 0

No. ofEmbryostaken.

B.

55coooCO

N

2215171217131313I I

886

11

88

1 0

. . .

Total No. 1 -of eggs J3 O°

Mgms.Urea perEmbryo.

C.

00003650004870-005000110000850021900273005201054007440-13501920-3040-3700-532058608580-909

1-500

Mgms. ]

Wet Weightof Embryo.

D.

0-304

!-4o}I-3°5r 2* ^11

2*020 12*QoO 1

4-382

JSS}4"9 45-0595-1065-9555-4"5-9285-I365-9655063

5-623...

>er cent.

Dry Weightof Embryo.

E.

6 0 841-6242-046-6136-015O-93'63-49

41-8

41-3

57-2

7O-46

ot^}?6"3

72-3166-40677253-6348-4035-i636-3729-42

31-79

DailyExcretion.

Daily abs.Increment

Mgms.

F.

00045

00046

00150

00275

0-033400500

005700112000660016200-05400272000510

. . .

...

Daily Ex-cretion Rate.

DailyIncrement

Rate.

G.

00001

00104

00125

00059

0016600070

005500046000960010800218002210

. . .

. . .

. . .

at which point it ceases to rise and becomes quite stationaryfor the whole of the rest of development. In other words,as far as the wet weight is concerned, the production orexcretion of urea is very intense after the fourth day andbefore the ninth day. At later periods although excretion ofurea is still going on, it only just succeeds in keeping abreastof the wet weight. It is certainly interesting that this intensiveperiod of urea production occurs exactly between the carbo-hydrate period and the period associated with the predominance

2 0 1

Joseph Needhamof fat metabolism. The effect might, of course, be due to manyother causes than to a specially intense combustion of proteinduring this period. For example, it might be due to a limitingfactor such as the incapacity of the embryonic liver at thisstage to turn urea into uric acid. That the developing livercan act in this way is probable from what we know of thefat-desaturation process in embryonic metabolism (30). If thiswas the case, however, an inflection in the curve of absolute

1-5-I-4--1-3-1-2-l- l -

2L0 7 -

&2: os-

0-4-05-

0-2-

01-

DAYSFIG. I.—The white squares represent mgms. ure» per cent, of wet weight of embryo ; the

dotted circles represent mgnu. per embryo, ia. absolute amounts of urea.

mgms. per cent, per embryo should appear at the time when theliver takes on this function, but this is not the case. It istrue that the liver may exert such an influence to a slightextent, but this does not seem to affect the urea curve as awhole. The activities of the enzyme arginase must also besuspect for the present, for we have unfortunately no data asto its presence or distribution in embryonic tissues. It isunlikely that any urea escapes into the yolk and the albumenthus vitiating the curve. Tests made on such fractions on theeighth and sixteenth days gave no indications of the presenceof urea there. I t would be strange if the embryonic excretapassed into its food.

2 0 2

The Energy-Sources in OntogenesisIn fig. 2 the urea content is seen related to the dry weight

of the embryo. Here the effect is the same but the shape ofthe curve quite different, for we have a peak at the ninth dayinstead of an inflection. After the ninth day has passed, theurea excretion far from keeping pace with the increase in drymatter quite fails to do so and drops away from it down toa possibly constant level of 30 mgms. per cent. But as beforeit is the ninth day which is prominent.

100-

10-

5

FIG. 2.—Mgms. urea per cent of dry weight of embryo.

The results of these experiments are very significant asregards the protein metabolism of the embryo, but at the sametime altogether incomplete. Until the full curve for uric acidis known, we shall not be in a position to discuss the significanceof the ninth day peak, nor shall we be able to calculate—mostimportant of all—whether the amount of protein combustionas indicated by the urea and uric acid excreted will accountfor the gradual lowering of the respiratory quotient from 0.9 to0.73. At the same time it is extremely interesting that thecurves for the urea content of the egg should show a peakand an inflection at the ninth day. If urea can here be regardedeven as a subsidiary straw to show which way the metabolicwind is blowing, the fact that its intensive production takesplace between the period of carbohydrate utilisation and that of

VOL. III.—NO. 3. 203 O 2

Joseph Needhamthe utilisation of fat is very important and must take its placebeside the other facts which were discussed earlier in thispaper. For their explanation a synoptic conception is required.

Summary.1. Attention is drawn to the numerous facts in the literature

of chemical embryology which point to a succession of energy-sources during ontogenesis, carbohydrate preceding protein andprotein preceding fat. This question is discussed.

2. The urea content of the hen's egg has been investigatedfrom the fourth to the nineteenth day of incubation. There isa period of intensive urea production from the fifth to the ninthday. After that point the excretion of urea fails to keep pacewith the growth and differentiation of the embryo.

3. It is pointed out that this period comes exactly betweenthe stage at which carbohydrate is known to be utilised as anenergy-source, and that at which the same may be said of fat.

My thanks are due to Professor Sir Frederick Hopkins forhis great interest and kindness, to Dr H. A. Murray and MissM. Stephenson for the stimulus of their conversation, and tothe Government Grant Committee of the Royal Society for agrant towards the expense of these researches.

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