Book - Chemical embryology 2-8 (1900)
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Needham J. Chemical Embryology Vol. 2. (1900)
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Chemical Embryology - Volume Two
Section 8 Carbohydrate Metabolism
8-1. General Observations on the Avian Egg
Our knowledge of the carbohydrate substances in the egg of the hen begins, where so much embryological history begins, with Wilham Harvey, who in the De Generatione Animalium says, "Egges after two or three daies incubation are even then sweeter rehshed than stale ones are. And after full fourteen daies (when the Chicken now beginneth to be downey and extendeth his Dominion over halfe the eggc and the yolke is almost still entire) I have boyled an egge till it was hard that so I might discerne the position of the chick more distinctly — and yet the yolke was as sweet and pleasant as that of a new laid Egge when it is likewise boyled to an induration". Compare with this, Fig. 275.
The subject of the carbohydrate metabolism of embryonic life is not a difficult one to deal with if a few initial propositions are kept in mind. Thus, as will appear later, glycogen cannot be considered as a representative carbohydrate, although some workers have assumed that it is. Then the earlier work has all to be judged in the light of the fact that copper reducing methods, so universally used for determining glucose quantitatively, give results which are far too high in the presence of protein breakdown products. The earlier work can accordingly be used only when there is reason to believe that peptides and amino-acids were excluded, as in the determination of free glucose after dialysis or precipitation of proteins, and not when the estimations were carried out on protein hydrolysates. Holden found that the Hagedorn-Jensen method, which involves the reduction of ferricyanide not of copper, was the most reliable, and the subsequent work of Pucher & Finch; Duggan & Scott; Fazekas; and Jonsell, Jorpes & Sikstrom has demonstrated the same thing. Passing now from questions of technique, it will be best to use our knowledge of the carbohydrate metabolism of the hen's egg as the skeleton on which to build up this chapter, for it is by far the best known case, and possesses all the important features, of embryonic carbohydrate metabolism.
8-2. Total Carbohydrate, Free Glucose and Glycogen
The first question which presents itself is naturally what happens to the total carbohydrate in the hen's egg during development, and how it is transferred to the embryo. The impossibility of estimating glucose by copper reduction methods in the presence of amino-acids as in protein hydrolysates makes the figures of Sakuragi for total carbohydrate uncertain, and the only available ones are those of Needham, who used the Hagedorn-Jensen method, after precipitating the hydrolysate with phosphotungstic acid. Hydrolysis was carried out with 5 per Days ^5
cent, hydrochloric acid for 5 pig. 264. The inset shows the early '
hours. In this way the amount portion of the curve enlarged.
of total carbohydrate, i.e. free carbohydrate plus carbohydrate combined with proteins plus glycogen, was determined each day during development (a) for the embryo and (b) for the rest of the egg. Fig. 264 shows the former values ; they rise as the embryo grows very regularly from i/ioth mgm. on the 3rd day to 70 mgm. at hatching. Similarly, Fig. 265 shows the behaviour of the total carbohydrate outside the embryo ; it first of all falls, then rises again somewhat, after which it falls again till the end of incubation.
Fig. 265.
Evidently by adding the data of Fig. 264 and Fig. 265 together, we obtain the amount of total carbohydrate in the whole egg on each day of development. This is shown in Fig. 266, from which it appears that at first the total carbohydrate in the whole egg falls, then rises again a little, and afterwards maintains a more or less horizontal course till hatching. In other words, the loss from the yolk and white after the loth day is practically compensated for by the gain of the embryo, but in the earner stages this is not onnL\ the case. Beside the main data on Fig. 266 are placed a few other points, some obtained by Sakuragi, using his own method and some which I obtained using that of Wood & Ost; both these depend on the reduction of copper, Fig. 266.
and gave higher results than
those of the Hagedorn-Jensen method, but they also show a constancy of total carbohydrate in the latter half of incubation.
Having ascertained the movements of the total glucose during the
we may proceed to consider the
mgms per egg
O Idzumi
• Pavy(whiteonly) e Bywaber8(white only) <D SatS ® Tomcta $ Gadaskin
( Vladimirovai.Schmidtt e Pennington 8 Hepburn &_ St. John V Kojo A Morner (white onJ_y)
Bernard C.
chick's development in its egg; movements of the various fractions into which it is divided.
It will first of all be of interest to compare the total carbohydrate of the remainder with the uncombined glucose there during the first half of development. Figures for the free sugar exist in some number in the literature, and, although all are " ' '"
derived from experiments in °' ' "
which copper reduction methd'ds were used, they are yet worthy of credence, because total hydrolysis with its production of amino"acids was not involved. Creatinine and glutathione would probably not be present in the protein-free filtrates, so that the objections against copper methods discussed above are not grave in the case of free sugar.
In Fig. 267 the figures of the various observers for the free glucose are summarised together. The sets of data are twelve in number, namely, those of Idzumi (Momose-Pavy method) , Sakuragi (MomosePavy method), Pavy (Pavy method), Bywaters (Pavy method), Sato (Schenck-Bertrand method), Tomita (Schenck-Bertrand method), Gadaskina (Galwialo method), Vladimirov & Schmidtt (HagedornTensen method), Pennington (method unknown), Hepburn & St John (FoHn-Wu method), Kojo (FehHng method) and Morner (FehHng method) ^ In addition, the figures of Claude Bernard & Dastre, the first of all, dating from 1879, ^re included. It will be admitted that Bernard's values are remarkably accurate in spite of the crude methods at his disposal. It is striking that with diverse methods the results are in such good agreement, and a glance at Fig. 267 convincingly shows that the free glucose in the yolk and white diminishes considerably during the first half of incubation.
In some cases investigators only give their results in terms of percentages. In order to reduce them to a common basis, therefore, it has to be assumed that they all worked with normal eggs under approximately the same conditions. From the data given in Table i, it may be assumed that of the weight of the entire egg at zero hour of development, 10-47 P^^" cent, is accounted for by the shell, 56-07 per cent, by the albumen, and 33-46 per cent, by the yolk. Using Murray's figure for the weight of an egg (average of over 500), namely, 57-8 gm., the white will weigh 32-04 gm. and the yolk 1 9' 33 gn^- The change in weight during early development due to loss of water by evaporation, assuming a constant humidity, can be read off on the graph given by Murray, The varying water-content of yolk and white due to the current of water yolkwards can be obtained from Fig. 225.
The free glucose beginning at a maximum of 200 mgm. per egg sinks more or less steadily till the loth day. An interesting point is the difference between the yolk and the white. Pavy and Bywaters only estimated the sugar in the albumen, and their points give a curve on a lower level than the whole egg curve, but roughly parallel with it. This might be taken to mean that there is not only a current of water but also a current of free glucose yolkwards, for by the 9th day the albumen has apparently lost all its free sugar, but the yolk has then lost only half its original amount. But Fig. 267 is misleading in that the values for the albumen are expressed as milligrams per egg, although the albumen does not alone account for anything like the entire Ggg, Fig. 268, in which the glucose is shown expressed as milligrams per cent, of yolk and white and plotted against time, is perhaps a better indication of what is going on. It demonstrates that, although both fractions of sugar fall markedly, there is a sort of cross-over in the middle of development, the yolk continuing at 75 mgm. per cent, and the white dwindhng away to nothing. If a passage of glucose into the yolk takes place, it must be by way of
1 See also the confirmatory data of Sagara (Schenck-Bertrand method) .
lGadaskina
Vlaci;mirov&,Schmidbb Tomiba
Sakuraqi
Yolk
Whlbe
Yolk
White
Yolk
White
Yolk
White
Yolk
Yolk&,Whibe(ld2umi)
Yolk8,Whibe(Sakuragi)
Yolk
White,
White (Mbrner)
Whibe(Pavy)
White {By waters)
the chick's blood-vessels. There is a piece of experimental evidence against it, for in 1921 Tomita injected glucose into the air-space of unincubated eggs. The idea had previously occurred to Pouchet & Beauregard, who, as far back as 1877, had injected | gm. of sterile "crystallised cane-sugar" into hen's eggs. Development was normal up to 13 days, but there was an odour of lactic fermentation. "Nous n'avons pu constater", they said, "la presence du sucre interverti dans I'albumine. £tait-il demeure dans son etat ou avait-il disparu soit dans le vitellus, soit consomme par I'embryon?" Tomita's researches were more enlightening:
Table 127.
1005
Amount of glucose injected
into the air-space before
incubation
( "^ ^
Mgm. Mgm. %
of glucose of egg-white
o o
1 00 390
50 200
200 800
Amount of alanine injected
into the air-spate before
incubation
Glucose found in
the egg on the 3rd
day of incubation
(mgm. %)
White 430 690 510 1080
Yolk
200 200 190 190
Mgm. of alanine
50
Mgm. %_ of egg-white
Glucose found in
the egg on the 3rd
day of incubation
(mgm. %)
White
410 400
Yolk
180 190
Tomita proved, in short, that the amount of glucose in the white can be raised on any one day by injecting a supply into it at the beginning of incubation, in spite of the factors which are leading to its disappearance, but that the amount of glucose in the yolk cannot be changed in this way. In passing, it may be noted that the addition of alanine to the egg-white before incubation had no effect on the glucose-content of white or yolk. It would therefore seem improbable that glucose normally passes into the yolk from the white.
We can now relate the curve for disappearance of free carbohydrate with the curves for other carbohydrate fractions. In Fig. 269 it is shown in relation with the curve for total carbohydrate of the nonembryonic part of the ^%g. The total carbohydrate of the remainder falls from zero hour till the 8th day, rises from then till the 1 1 th day, and thereafter falls steadily till the end of development. The free glucose also falls till the loth day, but not quite in the same manner as the total carbohydrate, for at the beginning its fall is slow, and thereafter rapid, while the total glucose first falls quickly, and slows down as time goes on. The latter part of the curve for free glucose, as shown in Fig. 269, is taken from the averaged estimations of Idzumi and Sakuragi, whose values closely agree.
Smoothed Curves
400
total carbohydrate In remainder
total carbohydraU in embryo
3bO
free carbohydrate in whole egg
300
- "Ovomucoid" in remainder
total cyclose in whole egg
250
/^-^^^ t
200
— ~. \
/ ^^\ x--^" °
150 100
V
'^ \ >v -"- -1
•"■••••....
^.
50 mgms
■__j:/
"" "■7"^!lW^'^---ir
frSc
n Days-^
5
10 15 20
ioo6
Before discussing Fig. 269 further, the glycogen in the whole egg and in the embryo must be considered. Glycogen has been estimated in the embryo by Idzumi and Murray, and their figures agree well^. They are shown in Fig. 270.
35 p o Glycogen in whole egg(ldzumi)
© Glycogen in whole eqgCSakuragi) e Glycogen in embryo (Murray) ® Glycogen in embryo(Sakuragi) Glycogen in remainder o
From these data it is simple to calculate the non-glycogen sugar in the embryo, and this is shown in Fig. 271 in its relation to the free sugar in the whole egg. It is seen that the rise in free sugar in the whole egg at the end of incubation goes almost exactly parallel with the rise of non-glycogen sugar in the embryo, maintaining a distance of about 60 mgm. from it. Thus at hatching there are about 50 mgm. of free sugar still remaining unabsorbed in the yolk-sac and presumably to be absorbed in the first few days of post-natal life.
Since the free sugar in the embryo (or, more properly, the nonglycogen sugar) rises parallel in the last 10 days with the free sugar in the egg as a whole, it is evident that the free sugar out- o side cannot be the source of the 1 200^ free sugar inside, or, if it is, it must be constantly replenished from some other kind of carbohydrate. During this time the total carbohydrate outside (see Fig. 265) is steadily falHng, and loses indeed in the last 10 days 160 mgm., during which time the embryo gains a total of 1 10 mgm. The difference must be either burned or transformed into some other substance, possibly cyclose.
1 Also subsequently by Vladimirov & Danilina, whose curve is almost exactly superimposable on that for the embryonic body in Fig. 270. Log. glycogen, they found, gives a straight line when plotted against log. age.
Free glucose in whole egg o Idzumi ® Sakuragi
a Averaged standard curve O Bernard i^Dastre in 1879 Non glycogen glucose in embryo o Needham
Days ^ 5
Fig. 271.
8-3. Ovomucoid and Combined Glucose
It is easy to calculate the amount of carbohydrate not present as glycogen or free glucose outside the embryo, for every day during development. This is graphically shown in Fig. 269. But such a calculation suffers from the fact that the non-glycogen sugar is assumed to be all free, which cannot be the case, but this error does not reach grave dimensions till the last 5 days of incubation, and as the correction would tend then to increase the free glucose outside the embryo it would tend also to decrease the ovomucoid glucose outside the embryo. We may therefore allow for the fact that the descent of this curve in Fig. 269 at the end of development is rather more precipitous than the graph makes it.
This curve, which, for want of a better name, we may call the "ovomucoid" curve, shows some interesting relationships. In the first place, its initial value, namely 133 mgm., is in fair agreement with the independent data of Komori, which have already been referred to (see p. 268). Komori prepared ovomucoid from fresh hen's eggs, obtaining from 333 gm. of albumen 4-8 gm. of ovomucoid. The processes were carried out as quantitatively as possible in order to get an idea of the concentration of the substance. This would mean 140 mgm. of ovomucoid glucose present at the beginning of development, which agrees very well with the 133 mgm. calculated from Needham's estimations by difference. This result gives us some confidence in interpreting the changes occurring in this fraction as changes in ovomucoid content.
What are these changes? As can be seen from Fig. 269, the ovomucoid curve falls until the 5th day is reached, after which point it rises to a peak on the loth day, thence to fall steadily till the time of hatching. The initial fall is of great interest in view of Komori's experiments in which he showed that Miiller & Masayama's egg "amylase" can very efficiently split off the sugar from ovomucoid. We may suppose that the heating of the egg at the beginning of incubation would set the enzyme in action, like the mechanism already suggested which controls yolk viscosity during the ist week (see p. 836). By the 20th day there are at most half-a-dozen milHgrams of ovomucoid left. All the carbohydrate outside the embryo at that time can be accounted for by glycogen and free glucose. From the fact that this peaked effect is found so markedly in the ovomucoid curve the conclusion might be drawn that ovomucoid is a more labile element in the raw materials of the embryo than has usuallybeen supposed. The work of Anson & Mirsky on another conjugated protein, haemoglobin, showed the ease with which the prosthetic group can be detached from and re-attached to the protein part of the molecule.
Komori gave other figures for the amounts of ovomucoid present during development, but expressed them as grams per cent, of dry weight of albumen, so that, although we know the rate at which the albumen is drying up, we cannot calculate his figures in milligrams absolute per egg because we do not know the relative weights of yolk and white. Sakuragi's figures for the same fraction are not valuable, being few in number and obtained by the use of doubtful precipitations prior to total hydrolysis and estimation by copper reduction. The only extensive work on the physiology ' of ovomucoid is that of By waters, who found that, between the ist and 1 8th day of development, the ratio of uncoagulable protein nitrogen to coagulable protein nitrogen in the egg-white was steady at 0-136, and therefore concluded that there was no preferential absorption of ovomucoid or ovoalbumen. This does not at first sight agree with the curve shown in Fig. 269. Two hypotheses are open: (i) that the curve for ovomucoid glucose calculated by difference does not accurately represent the ovomucoid glucose, or (2) that at varying times in development the amount of glucose combined in the ovomucoid molecule varies considerably. Both these seem possible. Bywaters found no change in the amount of sugar in the uncoagulable protein between the i st and the 1 8th day ; it remained constant at about 27 per cent., and, as the ratio of the two expressed as grams per 100 gm. egg-white was more or less constant (see Fig. 272 and Table 128), he considered that the carbohydrate radicle of ovomucoid was not split oflf before absorption. His methods are, however, not free from criticism, for he used the original method of Pavy without modification, and only hydrolysed the ovomucoid for 1-5 hours with 5 per cent, hydrochloric acid. Moreover his ratio varied from i-o to 2-4, which suggests that the hydrolysis was incomplete. It is significant that Bywaters' ovomucoid glucose values are highest between the 7th and 13th days, though their absolute values are at least 50 per cent, higher than mine. The second interpretation of the behaviour of the ovomucoid fraction receives some support from the work of Komori and of Levene & Rothen; for they have found the sugar to be combined with the protein as a polysaccharide. Further work on the constitution of ovomucoid and the changes which it undergoes during development is much needed.
Fig. 272.
Table 128. Bywaters' figures.
Ratio : Grams uncoagulable protein nitrogen per loo gm. eggwhite (wet) /grams coagulable protein nitrogen per lOO gm. egg-white (wet)
0-I7
0-I2 0-15
o-i6
0-I2
o-i6 0-14 o-ii
0-13
0-I2 0-15 O-IO
0-13
Day o
Ovomucoid in
grams nitrogen
per 100 gm.
egg-white
a
Ovomucoid glucose
in grams glucose
per 100 gm.
egg-white
j3 Ratio jS/<
0-27
0-42
1-6
0-26 0-32
0-40 0-33
1-5 i-o
0-53
0-76
1-4
0-65
I -30
2-0
0-49
o-gi
1-9
o-6o
1-43
2-4
0-I2
o-i8
Average ... 0-136
0-78
•29
In 1927, I estimated the amount of ovomucoid in the egg-white during incubation, and also the percentage of glucose in the ovomucoid molecule. The percentage of the whole egg accounted for by the white descended from 21 to o per cent. The ovomucoid isolated in milHgrams per Qgg fell from 1 28 to 4. Although a quantitative recovery of ovomucoid was not claimed (and indeed these figures are lower than those of Komori (see Fig. 273)), yet, as the same care was used throughout, it is probable that they have relative significance. When they are expressed as percentage of the wet weight of egg-white, it is interesting to note that the amount of ovomucoid
Fig. 273.
in 100 gm. of wet egg-white remains constant throughout development at about i gm. As far as these figures go, then, they confirm the observations of Bywaters, and indicate that there is no preferential absorption of ovomucoid from the white; although there may be from the yolk. This latter possibility is made likely by the fact that the hump on the ovomucoid-glucose curve comes just between the two times at 1 1° which the mucoprotein-glucose inside the embryo is high.
For the percentage of glucose in the ovomucoid molecule, the average value was 11-5, as has already been mentioned. This is probably more accurate than any other estimate, for it was obtained by the Hagedorn-Jensen method after phosphotungstic precipitation. But the curious thing about it is that, when the figures are plotted on a graph, as in Fig. 274, an upward trend is seen. No explanation has been found for this phenomenon. It is at any rate clear that there is no marked increase in the glucose content of ovomucoid at the period when it would be expected in the former of the two views outlined above.
Continuing the subject of ovomucoid-glucose, we may return to the discovery by Miiller & Masayama in 1 899 of an active starch-hy drolysing enzyme in the yolk of unincubated hen's eggs. This amylase would convert "under favourable conditions" 45 per cent, of a 3 per cent, starch solution into the soluble forms of dextrin and isomaltose in 24 hours at 37°. This put on a sure basis the earlier and rather doubtful results of Krukenberg, and was in turn confirmed by Diamare; Herlitzka; and Roger. Idzumi brought forward data which showed that the activity of the extra-embryonic amylase markedly increased as development proceeded, especially after the 15th day (rising from 40 to 640 units). These researches will be referred to in more detail in Section 14-7. Finally Komori demonstrated that most, if not all, of the glucose in the ovomucoid molecule could be liberated by incubation with amylase prepared from meal.
Fig. 274.
It remained to demonstrate that the egg itself, or certain parts of it, possessed the power of hydrolysing ovomucoid, and I made the requisite experiments in 1927, The ovomucoid preparation itself contained no free glucose, but when incubated alone about 2 mgm. were split off, perhaps because of the presence of minimal amounts of the enzyme. This auto-digestion effect was subtracted from the crude results. It was noted in the first place that the slight excess of glucose in the embryo preparations after standing at 37° was much more than accounted for by the correction mentioned above ; the effect of the egg-white was much reduced, but the yolk and the yolk-sac results still retained considerable magnitude. The enzyme ovomucoidase is thus not contained in the embryonic tissues themselves, and only to a negligible extent in the egg-white, but the yolk is very rich in it, and the blastoderm and yolk-sac also show a fair activity. Thus the embryo tests yield o per cent, of the theoretical, the white i -49, the yolk 31-2 and the yolk-sac 7-13. This conclusion is not affected by the varying amounts of solid material which must have been contained in the original samples, for the yolk retains its superiority even when this is allowed for.
These results fit in well with the rest of our knowledge. Roger, for instance, found more amylolytic activity in the yolk than in the white. Bywaters' conclusion that ovomucoid was not split up into protein and sugar before absorption only holds for the white and for that fraction alone the results do not conflict. The ovomucoid may be pictured passing from the white to the yolk by the vitelline vessels, and there being split up into free glucose and protein before absorption into the embryo. It is interesting that the embryo possesses no ovomucoidase, or very little; the hydrolysis must therefore be considered to take place outside it. Idzumi's observations on the increase of activity of the yolk amylase during development also fit in with the increasingly rapid disappearance of ovomucoid seen in Fig. 269. It would be desirable to extend these observations by investigating the kinetics of the enzyme, and by determining whether its activity remains the same in all fractions during development.
The question now arises as to the value of the ovomucoid to the embryo. It is probable that none of the ovomucoid is combusted during development, and the substance may therefore be considered of architectural rather than of energetical significance. This is in agreement with the main conclusion of Levene, who in his recent monograph ascribes to the mucoids in general a structural, cementing, and protecting value. From the ovomucoid curve in Fig. 269 we should expect to find some clues for its significance in the embryo about the 4th day and after the 1 1 th day, because at both these times active ovomucoid catabolism is going on. And indeed it can be said that at these two points the needs of two tissues are at a relevant stage, namely, the primitive connective tissue and the bones. In the formation of these mucoprotein will be required, as if for the interstices of the growing embryo.
Von Szily first described a cell-free fibrous connective tissue groundsubstance filling up all the cavities of the embryonic body in the early stages. This has some affinities with the "cardiac jelly" of Davis, and has recently been investigated by Baitsell, who has examined its properties with the aid of a micromanipulator. It appears to be secreted by the cells and provides them with a homogeneous matrix, a kind of natural culture medium, in which migration may take place if and when it may be necessary. As development proceeds the substance does not disappear, but becomes less and less important relatively to the body as a whole. The nearest equivalent to this ground-substance in later life is the Wharton's jelly of the umbilical cord and the vitreous humour of the eye. It is significant that both these tissues are known to be rich in substances of the mucoprotein type. It is probable that the decreasing importance of this primitive connective tissue may be related to the decreasing percentage of siUcon in the embryo (see Section 13), and perhaps it may have some relation to the fall in the water-content of the embryo throughout incubation (see p. 871). The amount of glucose in the embryo each day not free and not present as glycogen can easily be ascertained. Its gradual rise follows the growth of the organism. But if we express it in percentages of weight of embryo, we find that 100 gm. dry weight of embryo contain 2550 mgm. of "mucoprotein glucose" on the 5th day, but only 980 on the 15th. This result is an interesting comment on the suggestion that the importance of the primitive intercellular matrix will require a high proportion of mucoprotein.
The fall of ovomucoid in the latter half of incubation may be related to the process of bone formation, which, as von Baer established, proceeds at this time. Its calcium and phosphorus requirements have been studied in detail, and will be found discussed in Section 13-2, but very little attention has been paid to its need for osseomucoid. Even less is known about osseomucoid than about ovomucoid, but we may make a calculation which is suggestive. The amount of osseomucoid in fresh bone has not been accurately ascertained, and no estimation method has ever been devised for it. Hawk & Gies obtained 7 gm. of pure osseomucoid from 1 700 gm. of fresh bone (0-04 per cent.), but they were not trying to work quantitatively. On the other hand, we know from the analyses of von Bibra; Schrodt; Wildt; Morguhs; and Weiske that the organic matter in bone amounts to about 33 per cent, of the fresh weight. The nearest approximation to the amount of osseomucoid would seem to be onetenth of the organic substance. According to Tangl, the weight of the bones in the chick at hatching is 1-446 gm. dry weight, i.e. 3-616 gm. wet weight. This leads to an organic content of 1-205 gm. and about 120 mgm. of osseomucoid. Assuming its proportion of glucose to be 33 per cent., we get an equivalent in glucose of 40 mgm. Tangl's value for the weight of connecti\'e tissues at hatching is 0-405 gm. dry weight, or, assuming a water-content of 75 per cent., 1-615 gm. wet weight. The mucoprotein would here amount to about 60 mgm., and the corresponding glucose to 20 mgm. There would thus be 60 mgm. of mucoprotein-glucose present in the finished embryo. Since just under 200 mgm. of ovomucoid glucose have disappeared since the loth day, it is evident that the mucoprotein of the raw material has some other goal besides the mucoprotein of the completed article, but the temporal correlation holds good, for, just as during the period of importance of connective tissue there was catabolism of ovomucoid, so here in the period of growth of bone there is a similar effect.
The closely related ovomucoid of reptile eggs has been investigated by Takahashi on those of Thalassochelys corticata. During development the percentage of ovomucoid in the organic matter of the egg-white varies from 33 to 17 per cent. The ovomucoid is attacked by amylase and yields 6-2 per cent, glucose (notably less than avian ovomucoid). Takahashi was inclined to think, on the basis of rather few analyses, that the elementary composition of the substance changed as the embryo developed.
Before leaving Fig. 269, attention may be drawn to the figures for inositol-content of the whole egg, as determined by Needham's method. Owing to the inferior accuracy of this method, which yet is the only available one, no stress can be laid on the actual values, but there is a reciprocal correlation between the total cyclose and the total carbohydrate.
8-4. Carbohydrate and Fat
So far the analyses which have been described were done in all cases upon the embryo and the yolk and white separately. Sakuragi's important paper of 191 7 was based on analyses of the entire egg at different stages, and it is interesting to see how his results coincide with what has already been said. He adopted the plan of making a number of parallel observations with different methods, so that Fig. 275, which sums them up graphically, involves the following fractions :
A. (Hydrolysed residue after alcohol extraction) glucose of ovoalbumen, ovomucoid and glycogen.
B. (Alcohol extract after hydrolysis -D.), glucose of glycogen.
C. (Filtrate from protein coagulated by heat) free glucose.
D. (Alcohol extract before hydrolysis) free glucose.
E. (A. +B.+D.) total glucose.
F. (Hydrolysed residue after water extraction) glucose of proteins and of glycogen?
G. (Water extract) free glucose + ?
H. (Hydrolysed water extract with hydrochloric acid) free glucose + ?
I. (Total hydrolysis of whole egg with hydrochloric acid direct) total glucose.
J. (Ditto, a second time.)
K. (F.+H.) total glucose.
As Fig. 275 shows, Sakuragi did not observe any rise of total glucose in the egg as a whole between the 8th and i ith days. It is interesting that the free glucose determined in several different ways diminishes and then rises ; this agrees with a great deal of earlier and later work. The glycogen, estimated directly, rises all the time. The unknown hydrolysable carbohydrate which is found in the watery extract has the effect of diminishing but not of completely abolishing the fall and subsequent rise of the free glucose. Its presence warns us that there may be many important processes in the carbohydrate changes in the developing hen's egg which are entirely hidden from us at present. The decrease in glucose combined with protein found by Sakuragi equates with the similar decrease in Fig. 269, though the course taken is very different, and Sakuragi himself made no attempt to explain it.
A © (hydrolysed residue after alcohol exbractlon) i.e.,glucose of
ovoalbuman + ovomucoid + glycogen B • (alcohol extract after hydrolysis -d)
glucose of glycogen C O (filtrate from protein coagulated by heat) free glucose D D (alcohol extract before hydrolysis) free glucose E ® (a + B + D) total glucose F © (hydrolysed residue after water exbracbion)i.e,g!ucose
of ovoalbumen , ovomucoid fi^glycogen ? G O (water extract) free glucose + ? H © (hydrolysed water extract HCl) free glucose + ? I ♦ (total hydrolysis of whole egg HCLdirect) total glucose J A (ditto, a second time) KB (F + h) total glucose
Fig. 275.
It is important to remember that he was dealing with the egg as a whole, so that any diminution in a substance or fraction means its absolute disappearance; as regards ovomucoid glucose, this strongly substantiates the conclusions drawn above^.
Sakuragi considered that protein played no part as an energy source in the metabolism of the chick embryo. "It seems to be very interesting", he said, "that a small quantity of sugar is always kept undecomposed and moreover towards the end of foetal life some glycogen appears. The significance of this phenomenon may be interpreted as the necessity to convert the fat into sugar before it can be utilised as energy. . . . This is the reason why the egg contains a relatively high amount of sugar initially, sufficient to maintain the sugar balance until the organ capable of transferring fat into glucose has had time to develop."
However the kinematics of the inter-carbohydrate transformations may run, it is likely from Fig. 269 that the carbohydrate of the egg as a whole receives some reinforcement between the 8th and nth days. This increase amounts to about 90 mgm., so protein is at once ruled out as a source, because during the whole of development only 68 mgm. are lost, and most of this must be due to true protein catabolism. It is true that the peak in protein catabolism occurs at just the same time as the gain of carbohydrate, but as between the 8th and gth days the egg only loses i mgm. of protein while it gains 47 mgm. of carbohydrate this correlation is probably but a coincidence. We may safely conclude that the protein which is broken down is used for the production of energy, and being burnt away does not go to form that extra carbohydrate which appears in the middle of development.
The other possible source is fat, and here the position is a good deal more hopeful. As will be shown in Section ii-i, between the 7th and the 14th days there is a discrepancy between the fat lost as determined by the averaged chemical analyses and that determined by the carbon dioxide output on the supposition that all of it was due to fat, which is not true. More is lost than can be accounted for even on this assumption. "The figures of Bohr & Hasselbalch", I said in 1927, "would give an even worse divergence than those of Murray for they were lower than his. The explanation for this missing fat must be that during that period it is used for other purposes than combustion though there is, of course, the possibility that the estimations of fat are wrong." If now we suppose that the estimations were correct, and place beside the carbohydrate gained the fat missing each day, as is done in Fig. 276, we find that a correlation exists. Little account need be taken of the fact that the maximum of the carbohydrate gained is reached before the maximum of the fat lost, for the inferiority of methods, the comparative fewness of estimations, and the varying conditions of individual workers must all be borne in mind. Nor can stress be laid on the absolute magnitude of each quota, for an exact balance would show more carbohydrate gained than fat lost, since there is more carbon in a gram of fat than in a gram of carbohydrate. But it is striking that nowhere else in the development of the hen's egg can two such discrepancies be found, and that the two are approximately of the same order.
1 See also Sagara.
Fig. 276.
If then we accept the view that the increase in carbohydrate at this point is due to a transference from fat, there is one interesting corollary. It is that a transfer of a less oxygenated to a more oxygenated substance is taking place, and therefore that some oxygen would tend to be retained in the egg, and therefore that the respiratory quotient would tend to be lower than it theoretically should. If Fig. 1 44 be examined, it will be seen that there is a certain lowness of the experimentally determined respiratory quotient points as compared with those calculated from chemical analyses of food-stuff burnt. It is true that the passage from fat to carbohydrate, if it is of the order we are supposing, would not be large enough to make much difference, but it might well be one of several factors.
As is well known, the passage from fat to sugar is usually believed to be impossible in vivo, and it is certainly not supported by any unassailable piece of evidence drawn from experiments on the adult animal. In the starved dog the feeding of fat will not produce Hverglycogen, nor in the diabetic dog a rise in the G/N ratio, nor are any of the balance-sheet experiments, in which glucose seems to appear unaccountably and fat has to be postulated as its precursor, free from criticism. In the present case, moreover, it is true that a passage through the form of cyclose might account for the fall and the rise in Fig. 269, but no stress can be laid on the high absolute values for the inositol in White Leghorn eggs, and further, if the carbohydrate goal is abandoned some other objective must be found for the missing fat. As evidence for the possibility of the fat-carbohydrate route in vivo these arguments give support to the experiments of Furusawa and of Calvocriado. Furusawa considered that the liver is the organ responsible for converting fat into carbohydrate, and, if this is so, it is interesting to note that the liver-cells of the developing chick can make carbohydrate from fat before they can store carbohydrate when it is made.
We must now examine this property of storing carbohydrate, and to do so the metabolism of glycogen in the embryo must be dealt with more fully.
8-5. The Metabolism of Glycogen and the Transitory Liver
The increase of glycogen in the embryo has already been mentioned. But we also possess figures for the glycogen in the whole egg owing to the investigations of Idzumi and Sakuragi, arid, as they are quite concordant, confidence may be placed in them. They are depicted graphically in Fig. 270. By subtracting the glycogen in the embryo from the glycogen in the whole egg, we obtain that in the remainder, the figures for which appear as a dotted line in Fig. 270. By the end of development the glycogen in the embryo has only attained about a quarter of the total sugar in the embryo. It is interesting that Idzumi; Sakuragi; and Shaw all observed a great decrease of embryo glycogen during the process of hatching^, Idzumi to 10 and Sakuragi to 25 mgm. per embryo. They consider that this is related to the vigorous muscular movements which the embryo then makes for the first time, including the lung movements, which come into play as the animal lays aside its allantois. As may be seen from Fig. 270, the glycogen is at its highest outside the embryo about the 13th day; after that time it rapidly falls, and is, indeed, more or less the reciprocal of the glycogen inside. Evidently after the 13th day the glycogenic function is shifted from somewhere outside the embryo to somewhere inside.
1 Confirmed by Vladimirov & Danilina.
The conception of a late development of the glycogenic function of the liver is no new one. In 1858 Claude Bernard published his researches on mammalian embryos, in which he clearly showed that the glycogenic function later to be undertaken by the liver was, during the greater part of foetal life in mammals, carried out by the placenta. On p. 120 he wrote in a footnote, "Dans les oiseaux (poulet) j'ai constate, avant le developpement des cellules glycogenes du foie, I'existence de cellules glycogenes qui se developpent dans les parois du sac vitellin ; mais n'ayant pas pu suivre encore completement leurs evolutions, je traiterai ce sujet dans une autre communication, me bornant aujourd'hui a parler des mammiferes". This promised research was delayed for six years owing to Bernard's illness. On March 31, 1864, he deposited a "pH cachete" at the Academic des Sciences, in which he stated that he had shown the presence of glycogen in the blastoderm of the chick, and regarded it as comparable, from this point of view, with the placenta of mammals. Moreover, he had isolated the glycogen from the blastoderm and identified it chemically, obtaining from it alcohol and carbon dioxide by appropriate fermentation. In 1872 Bernard read a full account of subsequent experiments before the Academy, and caused his sealed communication to be opened and read. His own words may be quoted, "4 Juin i860. Sur un oeuf de poule du deuxieme au troisieme jour d'incubation, j'ai detache avec des ciseaux la membrane vitelline tout autour de I'area vasculosa; je I'ai enlevee avec des pinces de maniere a appliquer sa face exterieure contre une lame de verre. En examinant ensuite sous le microscope cette preparation, j'ai vu tres nettement des cellules glycogeniques et des granulations de glycogene qui prenaient une couleur rougeatre par la teinture d'lode acidulee avec I'acide acetique crystallisable". Bernard concluded that the blastoderm contained a notable store of glycogen for the needs of the embryo. He afterwards published a full account of his work in this field in his masterly Legons sur les phenomenes de la vie.
It will be convenient here to discuss birds and mammals indiscriminately with reference to what Claude Bernard called the phenomenon of the "foie transitoire " or temporary liver. Bernard described in detail the histological appearances of the placenta when stained to show glycogen, but he did not rely solely on histochemical observations, for he mentioned the results of several chemical experiments which led to the same conclusion. " II existe en effet, " said Bernard, "avant que le foie foetal puisse executer ses fonctions, un veritable organe hepatique placentaire qui produit la matiere glycogene." Bernard was for long puzzled by the fact that he could demonstrate this glycogenic function of the placenta with ease on guinea-pigs, rabbits, etc., but not on ruminants such as cows and sheep, but he eventually found that the reason for this was purely anatomical. In the case of the ruminants, the vascular and glandular components are quite separated, and, while the former continue to grow till term, the latter disappear by an atrophic degeneration. At birth, then, there may be very little left of the hepatically functioning part of the placenta. "One must add", said Bernard, "that all the time the amniotic placenta is increasing in size, the foetal liver possesses neither its adult functions nor its adult structure, while precisely from the moment that the foetal liver has attained an adult character and that its cells having acquired their definitive form, begin to secrete and store glycogen, the hepatic organ of the amnios begins to disappear," Bernard reported also that the cells of the skin, the intestinal mucosa, the mucous membranes of the respiratory and genito-urinary passages, the muscle-cells, etc., could be shown to contain supplies of glycogen in foetal life, and at a time when the liver was completely devoid of it. Glands and bony or nervous structures, however, were always free from it. This led to a series of researches on the glycogen content of tissues, mostly histochemical, which will be referred to later.
Immediately following Bernard's paper in the Annales des Sciences Naturelles, there is to be found a note by Serres entitled "Des corps glycogeniques dans la membrane ombilicale des oiseaux". Bernard's communication, he said, revealed to him the nature of those little glandular bodies which appear on the surface of the chick's blastoderm during incubation, and which he had figured previously without knowing what they were. "We see these little objects", he said, "from the twenty-fifth or thirtieth hour of incubation onwards. Their whitish colour suffices to distinguish them from the blood islands which have a reddish tint. At the thirty-fifth hour they become of a clear yellow colour and their increased size allows them to be more easily made out. . . . From the third to the sixth day their volume continues to increase but the proliferating arteries and veins partially hide them." About the 12th day they begin to disappear, and from histological considerations this time corresponds with the arrival of the foetal liver cells at a stage suitable for the storage of glycogen. "May we not say", continued Serres, "that there exists in the case of the chick a diffused hepatic organ, or a transitory liver, analogous to that which M. Claude Bernard has just demonstrated in the placenta of ruminants ? "
The work of Claude Bernard was for many years afterwards repeated and confirmed in a fragmentary manner. For the most part the work was histochemical, depending entirely on the iodine method. Thus Godet in 1877 described a "glycogen layer" in the rabbit's placenta. Langhans found none in the fully developed human placenta, though it was plentiful at eadier stages, as Merttens showed. Maximov, who made a very detailed study of the rabbit placenta, described the glycogen as increasing in amount up to a certain point, and then dying away. This was also observed by UleskoStroganova and by Chipman. who, however, differed greatly between themselves as to the actual situation of the glycogen. Chipman never found glycogen in the foetal part of the rabbit placenta. It appeared in the maternal part first on the 8th day, and increased rapidly in amount, reaching a maximum between the 12 th and i6th days. After that time it diminished steadily, and after the 22nd day there were only found small glycogen granules scattered in the midst of conglomerate masses of uni- and multinuclear cells, save in the zone of separation, where some cells remained distinct and retained their granules of glycogen. Glycogen was not found by Chipman in the foetal Hver of the rabbit before the 22nd day, after which it increased rapidly and steadily till birth. He did not observe any diminution after birth. Other histochemical workers were Rouget; Marchand; Brindeau; and Schonfeld, who all reported first an increase and then a decrease in the glycogen of the rabbit placenta. Jenkinson; Gierke; and Saake worked on the mouse placenta, and published similar results. Driessen and Barfurth investigated that of the guinea-pig, Kajimura that of the bat, and Happe; Plesch; and Todyo that of man, from this point of view. A particularly interesting result was that of Kiilz, who confirmed Bernard's finding that glycogen was present in the 2-day-old chick embryo, but could not confirm the presence of glycogen in the cicatricula. The histochemical workers did not confine their attention to the placenta, and several of them, especially Barfurth, stated that in the early stages of development little or no glycogen was to be found in the foetal liver.
These conclusions were criticised by Pfluger in 1903 with all his usual vigour. He maintained that the histochemical reaction was liable to be misleading, as in instances where it had been negative for early embryos he had succeeded in isolating glycogen from them, and even estimating it quantitatively. Pfluger did a good service in drawing attention to the inadequacy of histochemical methods, and soon a number of investigations appeared in which chemical analyses were made.
Barfurth himself, for instance, pubHshed figures for a rabbit embryo liver and placenta, which are included in Fig. 278. Butte reported a value of 8-7 gm. per cent, glycogen for the embryonic liver of the dog at term and of 0-42 for the ^ corresponding maternal liver. Paschutin found no glycogen in the livers of cow embryos 10, 14 and 21 cm. long, but isolated a certain amount from the liver of an embryo 40 cm. long. At birth McDonnell found 2 per cent. Demant found as much as 1 1 -4 per cent, in the liver of a dog at birth, and noted that the quantity steadily decreased for several days afterwards (see Fig. 277). The cat embryo at term, however, has not such large amounts of glycogen in its liver, according to von Wittich, who only found 0-23 per cent. It is difficult to assess these early papers.
For the human embryo, von Wittich found 0-24 per cent, glycogen at 5-5 months development. A. Cramer found an average of 1-45 per cent, at term for liver glycogen, and o-o8 per cent, for placenta glycogen. The latter figure was fixed at 0-52 by Moscati, who found more in a placenta of the 7th month, and more recently at i-o6 per cent, by Clogne, Welti & Pichon, though Bottazzi could hardly find enough to estimate. Perhaps his placentas were not quite fresh, for Moscati showed that on standing at room temperature their glycogen content diminishes, and reaches nil 24 hours after the cutting of the umbilical cord. Adamov found i per cent, in human foetal livers.
In 1907 Mendel & Leavenworth studied the occurrence of glycogen in the embryo pig. Their results will be referred to again; here the important point to note is that they could never find any in the liver before the stage of 23 cm. length, i.e. until the iioth day of development. Exactly analogous results were obtained by Zaretzki on the guinea-pig, from the liver of which no glycogen could be isolated till late in development.
Fig. 277.
Not until 1908, however, was the assumption of the glycogenic function by the liver put on a firm chemical basis. In that year Lochhead & Cramer made a very complete study of the movements of glycogen in the placenta and foetal liver of the rabbit.
It may be said at once that it led to a remarkable vindication of the views of Claude Bernard. Lochhead & Cramer appHed the Pfliiger method for glycogen to the embryos and placentas of rabbits. The maternal placenta of the rabbit can be divided into the following three parts : (a) that next the uterine muscle, which represents the plane of separation at the end of gestation, (b) an intermediate part, the region of the uterine sinuses, and (c) a part which extends up as a series of peninsulae between the foetal columns, which are analogous to the villi of the human placenta and were described by Chipman as ectodermic tubules with a plasmodial covering. Chipman found histochemically that there was a good deal of glycogen still left in (a) at the time of birth, but none in the zone of uterine sinuses and none in the region of peninsular projections, although both these were full of glycogen earlier. When the two parts of the placenta are pulled apart, the delicate peninsulae are left attached to the foetal placenta, so that the glycogen in the foetal part is really not foetal, but maternal. Thus by analysing the two parts of the placenta obtained by mechanical separation information was gained on the changes in glycogen of the two different parts of the maternal placenta. Lochhead & Cramer called the glycogen in the part of the placenta nearest the uterine wall the "distal glycogen", and that in the peninsulae and foetal placenta the "proximal glycogen". This latter portion would come principally from maternal tissue, though a small quota might be supplied from the true foetal part of the placenta. The percentage of distal glycogen they found to be quite comparable to that in the healthy adult liver, rising from the 14th day of development to the i8th, remaining constant from the i8th day to the 22nd, and then rapidly falling till birth. This is shown in Fig. 278. The proximal glycogen was very small in amount, reaching at its maximum only 1 1 mgm. per placenta as opposed to the 93 mgm. of the distal glycogen, and did not affect the curve of total glycogen per cent, of the placental weight, except at the time (between the i8th and 22nd days) when the maximal placental amounts are present. These facts are in exact agreement with the work of Chipman.
Fig. 278 should be compared with Fig. 270. It can be seen that they are fundamentally alike — in the case of the chick the rising embryo curve and the falling non-embryo curve cross at the 17th day of development; in the case of the rabbit the rising embryo curve and the falling placenta curve cross at the 27th day of development. It is interesting to enquire whether these >' cross-over points occur at equal -^ percentages of the whole developmental time, and it is easy to calculate what percentage of the total amount of glycogen in the system is at any given moment in the embryo and what percentage is in the adnexa. If this is done a graph is obtained like that in Fig. 279, from which it may be deduced that the mid-point in the assumption of the glycogenic function (the "cross-over point") by the embryonic liver occurs when 82 per cent, of the total development is achieved in the case of the chick and 91 per cent, in the case of the rabbit. Another interesting point which emerges from Fig. 278 is that the amounts of glycogen in the embryonic liver and the amounts in the placenta would not fall on a horizontal line if added together. This must mean either that some of the placental glycogen is destined for other organs of the embryo than the liver, or that a catabolism of carbohydrate as energy source is going on. The latter possibility fits in, of course, with what has already been said about the energy sources of mammalian embryos (see pp. 729 and 993).
"The most obvious phenomenon in the decidual cells of the rabbit is the presence of glycogen at a time when the foetal liver cells store
7 18 19 20 21 22 23 24 25 26 27 28 29 Days of development
278.
only the merest traces", said Lochhead & Cramer. They found that glycogen could not be detected histochemically in the foetal liver before the 22nd day. A variety of diets had no effect on the amount of glycogen in the placenta and embryo, a finding quite in harmony with other work, which demonstrates the remarkable independence of the reproductive system against external influences. "The constancy of the amount of glycogen deposited in the placenta and in the foetal tissues generally", said Lochhead & Cramer, "contrasts markedly with the fluctuations in the glycogen store of the adult liver, both in the normal and in the pregnant animal, and shows up again the autonomy of the glycogen metabolism of the foetus." Some interesting experiments on the effect of
, . 8 ph-rul Glycogen outside embryo
phloridzin were also made by ^ '-""'^1— Glycogen inside embryo
these workers. 0-6 gm. of phlo- 5 ^'^[~ Glycogen in embryonic llver /
ridzin was injected daily into "Sss- /'
the mother animals from the
8th day of gestation onwards,
the time when, according to °^^^|oRabbit 5
Chipman, glycogen first appears ^.^ ^ ^^
in the maternal placenta. The
result showed clearly that the placenta does not give up its glycogen
readily to the maternal organism; thus on the 23rd day of gestation,
a normal animal would have 4-5 per cent, of glycogen in its placenta
and a phloridzinised one 4-24 per cent., or again 2-56 and 2-68 per
cent, respectively. In some cases, however, there was an interference
with growth, and the embryos were stunted; when this was so, the
glycogen percentages were less than normal. As for the foetal liver
after treatment with phloridzin, it had a lower glycogen content
than normal, but yet much higher than the corresponding maternal
liver.
Since Lochhead & Cramer's classical investigations, the general inter-relations between the liver and the placenta laid down by them have been confirmed by a number of workers. Thus Clogne, Welti & Pichon found in human embryos that the liver glycogen increased from i-8 gm. per cent, dry weight at the 3rd month to 29-5 gm. per cent, at the gth month, while the placenta glycogen correspondingly decreased from 2-75 at the 3rd to 1-05 at the gth month. For this last value, Higuchi got a lower figure — 0-032. Again, Loveland & Maurer found 1 00 mgm. of glycogen in rabbit placentas of 22 days' gestation and less than 25 mgm. at 32 days, and their histological checking fits in down to the last detail with what has been said above. Correspondingly Snyder & Hoskins reported that the glycogen of the rabbit foetal liver rose from a trace to 40 mgm. per gram — as much as the adult possesses — while the glycogen of the foetal body increased fivefold.
Interesting experiments have also been made by Huggett who has given attention to the factors which modify the amount of placental glycogen. These turned out to be few, for the percentage of glycogen in the placenta of the rabbit was unchanged by starvation, by carbohydrate feeding, by injections of carbohydrates, or by injections of hormones (adrenalin, thyroxin). Repeated injections of large doses of insulin did succeed in slightly lowering the percentage, and as phloridzin had already been shown by Lochhead & Cramer to have a similar effect, Huggett concluded that the only influences having any marked action on the glycogen of the maternal placenta were the profound disturbances of metabolism induced by massive insulin doses, phloridzin, and semi-pathological changes such as those induced by ether, amytal, and tetrahydro-j3-naphthylamine^. Wertheimer has also made similar observations. All the glycogen of the maternal organism can be mobilised in the rat or guinea-pig by the action of cold and adrenalin, but the glycogen in the foetus remains untouched. This immunity persists for some time after birth, newborn animals requiring huge doses of adrenalin to shift any glycogen. It was significant that neoplasms also remained uninfluenced by treatment affecting the carbohydrate stores of the rest of the animal, and Wertheimer in other experiments (see Appendix 11) found that amphibian ovarial eggs possessed a like independence.
We may now return to the development of the chick embryo, and unravel the mechanism of its carbohydrate metabolism further. Fig. 270 shows that the nth day of incubation may be fixed on as being the point in ontogeny at which the glycogen storage in the embryonic body begins to become notable, when considered in absolute terms, or at which it is increasing more rapidly, if considered in terms of per cent, dry weight (see Fig. 280 taken from Murray^) . Both Mellanby and Schmalhausen have weighed the hver at different ages in the embryonic chick, but the 1 1 th day is not associated with any specially marked change in weight. Nevertheless, that it is the embryonic liver which accounts mainly for the results obtained on the entire embryo appears strikingly from the careful histochemical work of Potvin & Aron, who in 1927 examined the liver of the chick during each day of development, and could find no traces of glycogen in it until the nth or 12th day. After the 14th day they reported that its increase was very rapid, a finding in exact agreement with the chemical results of Murray and Sakuragi^, and a confirmation of some forgotten observations of Claude Bernard. Another rather abrupt change in the Hver of the embryo chick was revealed by the investigations of Heaton, who observed a change in the biological properties of the liver cells on the 1 1 th day. Before that time, its cells in tissue culture grow like epithelial cells (having arisen, of course, as a diverticulum from the gut), but after that time they grow like fibroblasts. Nor is this merely a morphological difference, for after the 1 1 th day their growth is inhibited by yeast extract, just as that of fibroblasts always is, but this is not the case earlier. The change takes place regularly between the i ith and 12th days, i.e. just about the time when the curve for glycogen in the embryo shown in Fig. 280 inflects and rises sharply. In this connection it is significant that Holton found that the liver will not grow in chorio-allantoic grafts after the nth day. Nordmann has studied the metabolic behaviour of explanted liver-cells. He states that glycogen (observed histochemically) is synthesised by all stages from the gth day onwards. The earliest ones (gth day) showed the presence of glycogen very soon after explantation and retained it for nearly a fortnight in culture, the later ones (i ith or 12th day) retained it only for about 5 days. This synthesis of glycogen seemed to be independent of the constitution of the medium.
^ Conversely, placental glycogen cannot be increased by alimentary hyperglycaemia (Runge & Hartmann; Kessler).
Fig. 280.
^ Vladimirov & Danilina's curve rises similarly but somewhat higher throughout. - And of Vladimirov, who finds that between the 14th and 20th day of development the liver glycogen accounts for between 35 and 40 per cent, of the whole.
Serres' histological observations, illustrated in the Archives du Museum d'Histoire Naturelle, have since been confirmed by J. T. Wilson and by H. J. Allen. Allen, who seems to have been ignorant of the pioneer work of Serres, made a histochemical study of the glycogen in the yolk-sac of the chick at various stages. She found it to be present from the earliest time onwards, distributed all over the vascular area in the form of mahogany-coloured masses scattered among the cells. The head ectoderm, the heart, and the myotomes acquired glycogen very early also. "Apparently the yolk-sac", said Allen, "furnishes a way-station in which carbohydrates are stored as glycogen on their way from the yolk to the embryonic tissue."
Since the chick and the rabbit both exhibit the phenomenon of the "foie transitoire", the practice may be very general. It has been shown to take place in the ovo-viviparous selachian Mustelus vulgaris by Blanchard, who reported that the vitelline membrane contained abundant stores of glycogen. His methods were histochemical and the research was never published in detail.
The transitory liver may be regarded as an excellent instance of those special functions of embryonic life which will be discussed in the Epilegomena.
8-6. Free Glucose, Glycogen and Insulin in the Embryonic Body
It will be convenient now, before proceeding further, to ask what happens to the free carbohydrate of the embryonic body, for so far this important fraction has not been discussed at all. Only one set of measurements exists, ^ those of Needham, but these were done on a very large number of embryos with the Hagedorn-Jensen method, and some reliance may be placed on them. The curve for absolute milligrams of free glucose per embryo, after having been suitably corrected for the presence of creatinine on the basis of the factor introduced by Holmes & Holmes (that 8 mgm. of creatinine affect the Hagedorn-Jensen reagents to the same extent as i mgm. of glucose), naturally rose, keeping pace with the growth in size of the embryo. It is shown in Fig. 281 . It will be remembered that the total glucose in the embryo also rose steadily with the increasing size, as is shown by Fig. 264, but, if the two are compared, it will be seen that the shape of the curve is not exactly the same in the two cases, so that, when the two are related by expressing the free glucose in percentage of the total glucose, a peaked curve emerges (see Fig. 284).
For the moment, however, attention may be directed to the relation between free and total glucose and wet and dry weight, as shown in Fig. 282 and Fig. 283.
Fig. 281.
Fig. 282.
We see that 100 gm. of embryo (wet weight) contain on the 5th day of development 160 mgm. of total carbohydrate and 8 of free carbohydrate. The former falls to a level of about 100 and subsequently rises to over 300, the latter rises all the time in an S-shaped curve to about 48 mgm. This would seem to indicate that the carbohydrate present in the embryo on the 4th and 5th days, at which time there is more in proportion than at any subsequent period, is not in the form of free glucose, and as it cannot be glycogen it must be sugar in some unidentified combined form.
The dry weight data bear out this view even better. On the 5th day 100 gm. of dry embryo contain 3000 mgm. of total carbohydrate, about 100 mgm. of free carbohydrate, and about the same amount of glycogen. After that point the total carbohydrate continuously falls, reaching a value of 1750 on the i6th day, while the free carbohydrate continuously rises, its highest point being reached on the 1 1 th day with a ^°°° value of 360 mgm., after which i it falls, but not below 220 mgm. e^°°° The fact that the total glucose | falls, while the free glucose "5,2000 rises, is of some interest. The 2 presence of such a large pro- 0,000 portion of glucose not free and 5 ^ not in the form of glycogen a may be correlated with certain histological facts which have been known for some time, and which have already been referred to (see p. 566).
What happens to the concentration of the various carbohydrate fractions in the water of the embryonic body? It was pointed out above that when total protein, total carbohydrate and total fat were expressed as grams per 100 gm. of water in the embryonic body, the protein and the fat start at a very low level (owing to the great wetness of the earhest stages) and rise steadily, while the carbohydrate begins fairly high, falls to a minimum on the 8th day, and thereafter rises with the others (see Fig. 221). This was interpreted as illustrating the importance of carbohydrate as an architectural material in the youngest stages, an importance which was, however, only transitory, and gave way before development was half complete to the predominance of protein and fat found in the adult. The data for the free glucose made it possible to determine to which of the carbohydrate fractions the total carbohydrate concentration curve owed its preliminary peak. Without doubt it is the great amount of mucoprotein glucose present in the embryo in the initial stages which causes this effect.
Fig. 283.
Since we know the glycogen present in the embryo each day, a glycogen/glucose ratio can be constructed. As Fig. 285 shows, it remains steady till the middle of incubation, after which it rather suddenly rises, and acquires a new steady value. The point which is important here is the peak in the curve representing the free glucose in percentage of the total glucose, and, at exactly the same time, the rapid change in the glycogen/glucose ratio. Why should there be a higher proportion of free glucose in the embryo then than at any other time ? The answer must surely be that the free glucose goes on increasing until a point is reached at which the production of insulin overhauls it and a control of the proportion of free and combined sugar can be attained. Fig. 285 shows diagrammatically the part played by the pancreas in this mechanism. In early embryonic life there are one and a half times as much glucose as glycogen, but after the critical i ith day there are one and a half times as much glycogen as glucose. In this way the embryo rather quickly approximates to the adult condition, once the time has come for it to do so, for a preponderance of glycogen over free glucose may roughly be taken as characteristic of the mature animal. Another instance of such a comparatively rapid passage from the embryonic to the adult condition will be seen in the nitrogen partition in the urine of the chick (Section 9-5).
Fig. 285.
It is interesting to note that the effect of insuHn on the free glucose curve seen in Fig. 284 is a phenomenon revealed in the intact organism following its usual courses and not subject to the abnormaUties necessarily consequent upon depancreatisation or other experimental treatment. Another point is that the appearance of insulin occurs in that portion of embryonic life in which development is irrevocably determined and self-differentiation is going on. During the early stages, when a great degree of regulation is possible, the embryo has no insulin, and the hormone only arises after chemodifferentiation has taken place and the fate of every cell is finally determined. As will appear in Section 15, good evidence also exists that this applies to other hormones besides insulin, and probably to all of them.
Hanan demonstrated that insulin hypoglycaemia can be produced during the last week of development. Taking 14-16-day White Leghorn embryos, he injected insulin into the air-sac, and then, withdrawing blood from the allantoic vein at its bifurcation just below the air-sac, estimated the blood sugar by the Hagedorn-Jensen method. From the normal value of 209-296 mgm. per cent, a marked lowering could be observed. If glucose was injected into the air-sac instead of insuHn, the blood sugar rose, and it could also be made to rise by bleeding the egg. Riddle's finding that adult birds will survive a dose of insulin thirty times as strong as that which would kill a rabbit was extended by Hanan to this later period of incubation.
In 1922 Aron, as the result of comparative studies on the embryos of the sheep, guinea-pig, pig, and man, made the generalisation that the glycogenic function of the liver (as judged by the accumulation of glycogen within it) always occurred at the moment when the interstitial portion of the pancreas was taking on its adult histological appearance. His experiments did not include the determination of the glycogen content of the placenta, but he analysed the foetal liver at different stages of development, and obtained the curves shown in Fig. 286. "The glycogenic function of the liver", said Aron, "manifests itself at a fixed time in ontogeny. Its installation varies in different species, for with the sheep it appears early, at the end of the second month of gestation, but does not rise much till the end of the fourth month. With the pig it appears suddenly at the beginning of the last fortnight of gestation. With the guinea-pig it appears at the 40th day of gestation but does not rise very fast." Aron was not inclined to accept the simple hypothesis of Claude Bernard that the glycogenic function is taken on by the liver as soon as the cells are morphologically ready to receive it. He regarded it as much more likely that the passage of this function from placenta to liver was under the control of an endocrine agency, and was thus led to examine the behaviour of the pancreas. Laguesse had already pointed out that the appearance of the pancreatic islets in early embryonic life was quite different from that which they presented later, and Aron extended his work by showing histologically that the second generation of islets, i.e. the islets of Langerhans, appeared rather rapidly out of the islets of Laguesse just about the time when the glycogenic function was installing itself in the liver. This relationship held quite rigorously as between the different animals ; thus in the sheep the islets of Langerhans appeared first about the 50th day, while in the pig they did not appear till the looth day. The significance of these findings can be seen from Fig. 286. Aron also brought various lines of evidence together to show that the Laguesse islets produce no insulin, as those of Langerhans do. The transformation of the former into the latter may be very sudden. "Cette veritable explosion endocrinienne ", said Aron, "pent se declancher un peu plus tot ou un pen plus tard selon les cas. Quoi qu'il en soit, on observe constamment une coincidence frappante entre la presence de nombreux ilots de Langerhans dans le pancreas, et le depot dans la foie d'une notable quantite de glycogene." Even in the case of man, where Aron only worked out the appearance of the Langerhans islets, the relation holds, for the change occurred at the beginning of the 4th month of gestation, and from the work of Livini we know that no appreciable quantity of glycogen can be shown to be present in the hver before that time. Gierke and Lubarsch both made similar statements about the liver in early pig embryos. Thus the biliary always appears in ontogeny before the glycogenic function of the liver.
Fig. 286.
In 1928 Aron extended these conceptions to the amphibian embryo and larva. Glycogen was determined by histochemical methods in the livers of Rana temporaria, Rana esculenta and Bufo vulgaris. Aron found, just as Claude Bernard had found long before, that none is present before the appearance of the hind limb buds, i.e. before the disappearance of the yolk. Complete ablation of the pancreas in early stages altogether prevented the appearance of the glycogenic function, so Aron naturally concluded that the mechanism of its control in amphibia was similar to that in mammals. This would mean that the chemical regulation of the embryonic and yolk-sac period in the frog is carried on without the aid of insulin. Aron has suggested various further mechanisms of control involving the action of the thyroid, but these are not at present very certain.
Judging from the work of Goldfederova, the increase in liver glycogen, when it does come, must be very sudden, for she obtained the following figures :
Stage Hind limb buds visible Mobile hind limbs and tails Front limb buds visible Resorption of tail Completely metamorphosed
Claude Bernard himself went further afield than to the amphibia, for in studying the embryos of molluscs, especially the common oyster, he observed that the cushion or disc which carries their ciHa and makes them mobile was very rich in glycogen (histochemically) . As the disc subsequently falls off when the embryos become sessile, Bernard felt justified in seeing in it an analogy with the placenta of mammals as regards glycogen storage.
In other researches Aron, Stiilz & Simon carried out the experiment of Carlson & Drennan at various stages of gestation in the dog, i.e. they depancreatised the mother, and observed whether there was any evidence of protection from hyperglycaemia due to
Glycogen % Liver
vitt weight Muscles
II-9
0-05
9-7
o-i6
9-6
0-20
10-7 8-6
0-35
insulin from the foetal pancreas. Such experiments are open to various criticisms on grounds of technique, and in Section 21 '8 we shall examine them in more detail. They found that up to the 7th week there was no protection but that it was demonstrable afterwards. This agreed chronologically with the time at which the transformation of Laguesse islets into Langerhans islets was going on, and suppHed more evidence that the former were incapable of secreting insulin. Then Potvin & Aron investigated the islet tissue in the pancreas of the chick. They found that the first islets appear on the 8th day of development, and for a day or two those of Laguesse predominate, but these soon give place to Langerhans islets, so that by the 15th day there are none of the former left. It is impossible not to be struck with the correlation between these histological and physiological data, and the biochemical evidence of Figs. 284 and 285.
8-7. General Scheme of Carbohydrate Metabolism in the Avian Egg
A general scheme of carbohydrate metabolism in the developing chick may therefore be provisionally summarised as follows:
{a) Zero hour of development till the yth day. The carbohydrate in the embryo at the earliest stages, i.e. about the time when the yolk is first completely enclosed by the blastoderm, is to a very large extent composed of glucose in combination with protein. Glycogen increases outside the embryo, being stored in the yolk-sac and parts of the blastoderm. Free sugar increases steadily within the embryo both absolutely and relatively, being uncontrolled by the pancreatic hormone. The liver and pancreas arise as epithelial buds from the wall of the duodenum on the 3rd day. Glucose combustion predominates.
{h) Eighth to nth day. The importance of the mucoprotein fraction of the glucose in the embryo diminishes, but the free glucose continues to rise proportionately, as does the glycogen in the yolk-sac and blastoderm. At this time 90 per cent, of the egg's glycogen is outside the embryo. The liver and pancreas change their growth, the former increasing in bulk and the latter developing the islets of Laguesse. By the loth day the islets of Langerhans are in evidence, and insulin is being secreted in increasing amounts. At this time also a certain extra quantity of fat enters the embryo from the yolk, and is there transformed into carbohydrate.
(c) Eleventh day. The insulin about this time attains such concentration or activity that it is able to stop the relative increase of free glucose, which now passes through its peak. Perhaps the appearance of insulin is sudden, for Pucher & Hanan could not find any in the embryo until the i ith day. The Hver cells, changing the character of their growth, prepare to receive stores of glycogen, and from this point onward do so (Murray; Sakuragi; Potvin & Aron).
{d) Eleventh day till the end of embryonic development. The control of the free glucose by the insulin continues, so that as per cent, of the total glucose it declines. Synchronously with this process the glycogen of the embryonic liver increases steadily in amount, its origin being partly the glycogen of the transitory liver in the blastoderm and partly the diminishing free glucose. In other tissues also, e.g. the intestinal walls (Maruyama) glycogen steadily increases. The carbohydrate systems in the chick are now fully sensitive to the action of insulin.
The concomitant events outside the embryo may be traced by the curves given above. The most remarkable items seem to be that, when the free carbohydrate is at a maximum in the embryo, it is at a minimum outside, and that the periods of maximum importance of mucoprotein glucose inside the embryo coincide with the periods of maximum catabolism of ovomucoid outside.
8-8. Embryonic Tissue Glycogen
A good deal has been said already about the glycogen in the foetal liver at different stages, and the question now arises as to what happens to the glycogen in the rest of the body and its different parts. Fig. 270, embodying the data of Murray and Sakuragi, demonstrates that in the case of the chick the glycogen in the embryonic body as a whole rises in a regular curve both absolutely and relatively. In the last half of last century, much importance was for some reason attached to the embryonic glycogen, and it was thought that this substance was in some mysterious way connected with growth and differentiation. This belief died out when it came to be found that glycogen is not present in embryonic tissues to a greater extent than in adult ones. Fig. 287 shows the combined data of various investigators, the abscissa being in all cases conception age. Gage claimed to have seen glycogen histochemically in pig embryo brains, but Mendel & Leavenworth could not find any there, thus confirming Bernard, although they readily obtained it from the
Glycogen
1.2
r 0-7
1«1
0.6
1-0
0-9
. 0.5
0-8
0-4
0'7
0-6
0.3
0-5
"0.2
0-4
0.1
0-3
0-2
© © ©
O Rabbit, Lochhead.!LCramer(liver excludecl)% web wb. Chick, Murray (liver included) % dry wb. I
© Wliole Pfg , Mendel S^, Leavenworth °/o web wb. © Pig muscles, Mendel <So Leavenworth © Pig skelebon, Mendel <S^ Leavenworth -^Man muscle, von.Wibbich /^ web wb. n Cow, Collip
Dog, Liesenfeld Dahmen S(^Junkersdorf
Fig. 287.
muscles and skeleton. It is evident from Fig. 287 that the embryonic tissues show no special richness in glycogen.
In spite of these chemical facts an enormous quantity of labour has been expended by investigators who have charted out histochemically the various regions of the embryo according to the depth of colour obtained with iodine and other reagents. The two most outstanding surveys of this kind are those of Creighton and of Sundberg, in whose papers will be found lists of tissues and organs microscopically rich and poor in glycogen respectively. Creighton introduced the theory that glycogen "acts in the embryo as the precursor or deputy of haemoglobin until such time as the vascularity of the part is sufficiently advanced, and in other cases as the substitute of haemoglobin from first to last, i.e. in those tissues which are built up in whole or in part without the direct access of blood". Creighton also thought that "the cartilages which are destined to continue throughout life as cartilages have little or no glycogen in the foetal period, but those which later will ossify have plenty and it usually appears in the spots which afterwards become ossification centres".
Nobody now accepts Greighton's views and the attribution of any special embryological importance to glycogen is superfluous. While it may be useful to know the histological distribution of glycogen in the embryo, at present little physico-chemical meaning can be attached to most of this work. Investigators continue to labour along these lines, however, e.g. Ellis; Gragert; Glinka; Gierke; Gage; Lubarsch; Togari; Jordan. Livini has published a series of papers on the glycogen distribution in human embryos. None can be demonstrated in the liver till the end of the 2nd month. The muscles, lungs, skeleton and epithelial tissues begin then to acquire it; the pancreas, salivary glands, thyroid, parathyroid, thymus, suprarenal medulla, kidneys, smooth muscles, testes, ovaries, etc., have very little, and it appears irregularly, while the central nervous system, the suprarenal cortex and the retina never have any at all. Livini found that, as the liver glycogen rises towards the end of gestation, it falls in the organs of the second class, so that an additional source, other than the placenta, may be envisaged. All Livini's work is histochemical. It is clear that the glycogen of the body as a whole rises during embryonic life (Murray for the chick, Lochhead & Cramer for the rabbit, Mendel & Leavenworth for the pig, and Aron for the cow and the sheep) . The only contradictory piece of evidence is contained in a dissertation by Kistiakovski, who stated that the quantity of glycogen in embryo cows and sheep diminished gradually until birth. This publication is not available in England, but it can probably be disregarded in view of the consensus of opinion.
Some other experiments on glycogen in embryonic life are worth mentioning. Driessen's histological work showed that the early ovum of the rabbit, the mouse, and man (from the 3rd to the 6th week in the latter case) is surrounded by a layer of cells very rich in glycogen. This is probably significant for the nourishment of the embryo, and brings to mind the observation of Zavattari that the test cells of ascidian eggs are full of glycogen granules also. M. R. Lewis studied the cells of early embryos of the chick and the minnow in tissue cultures. Glycogen was always present according to histochemical test in the latter case, but only for the first 48 hours of development in the former. This is in agreement with Kiilz's work already mentioned, where 5000 6-hour cicatriculae were taken and worked up for glycogen.
Another analogous line of investigation is that of Vastarini-Cresi, who has studied histochemically the appearance and distribution of glycogen in the chick embryo. The first traces appear, according to him, in the heart at the 2nd day of development, i.e. at the beginning of pulsation. He considers that the canaHsation of vessels and spaces is specially associated with glycogen metabolism. He divides organogenesis into three phases: (i) Active cell multiplication in all directions to form a bud; here no glycogen can be found in them. (2) A period of glycogenic infiltration followed by one of canalisation or cellular dissolution. The inside cells become so full of glycogen that they cytolyse, and so form cavities. (3) Removal of the glycogen to other places.
8-9. Embryonic Blood sugar
The question of embryonic blood sugar and what happens to it must now be taken up. As long ago as 1875 Moriggia gave figures for foetal blood sugar in many animals.
Hanan's figure for the embryonic blood sugar of the chick at the 15th day is not in agreement with the later one of Vladimirov & Schmidttj who also used the Hagedorn-Jensen method, but probably worked on chicks of a different breed. They found a constancy until just before hatching, and considered that from the nth day onwards insulin was an effective agent in regulating the blood sugar leveP (see
1 Vladimirov has also studied the effect of adrenalin, asphyxia, etc. on the embryonic blood sugar of the chick. The results of Riddle & Honeywell agree with Vladimirov & Schmidtt rather than with Hanan, for they found the blood sugar of the embryo to be always less than that of the adult (3 species of pigeons) .
Fig. 288). The blood sugar, however, is not a large enough factor to affect to any great extent the free glucose of the embryo as a whole, as a rough calculation demonstrates. Assuming 230 mgm. per cent, for the 20th day, the amount of blood in the embryo would be
1-54 gm. (from the formula of Dreyer & Ray, i.e. 5 = -^, takmg
the mouse value (6-7) for K as the mammal nearest in weight to the hatching chick). This would give 3-53 mgm. of blood sugar, which, although a high estimate, is only 15 to 20 per cent, of all the free sugar in the embryo.
The results which have been obtained on mammalian embryos are sHghtly more coherent.
Aron investigated the blood 99nU oChick.viadimirov^Schr sugar of embryos of the guinea-pig, rabbit, dog, cow and pig, using the FontesThivolle method and accumulating the results shown graphically in Fig. 289. Although the points arrange themselves roughly along definite curves, Aron did not treat them as if this was so, but simply averaged out the data, and concluded that for each species of embryo there was a characteristic blood sugar level which did not change appreciably throughout intra-uterine Hfe. The values thus calculated were as follows :
Fig. 288.
Blood sugar in mgm. %
Species
Foetal
Maternal
Guinea-pig
60
107
Rabbit
It
125
Dog
no
Cow
no
100
Pig
139
100
Thus in some cases the glucose concentration in the foetal blood was higher, in other cases it was lower, than in the maternal blood. For comparison, the following values of other observers may be advantageously placed here.
Blood sugar in mgm. %
Investigator Olow Rowley Hellmuth ...
Morriss Bergsma
Species Man Man Man
Man Man
Foetal
80
90
Maternal
Foetal blood sugar always lower than maternal by 9-84 mgm. %
115 132
Foetal blood sugar the same as maternal at birth
These will be dealt with in detail in the Section on placental permeability. At present we must neglect the apparently regular changes in Aron's data and assume, with him, that the embryo is able to regulate its own blood sugar, for that was the point on which he laid special emphasis. "Le miheu interieur du foetus", he said, "reste, au point de vue de sa teneur en glucose, independant du miheu interieur de la mere. Tout se passe done comme si le foetus reglait sa propre glycemie."
As we have already seen, Aron established the fact that the appearance of the islets of Langerhans in the foetal pancreas and the installation of the glycogenic function of the liver '^* ^ ^'
are synchronous events in the life of the embryo. Aron now found, working with dogs and cats, that, although the islets of Langerhans appear in the former case not till after the 7th week of gestation and in the latter case well before this time, their appearance was always marked by a greater independence of the foetal circulation. Before their appearance, the removal of the maternal pancreas led to an elevation of the maternal blood sugar which was reflected very closely by a rise in the foetal blood sugar, but afterwards, this correspondence was not nearly so marked. Thus at the 6th week in the dog, after removal of the maternal pancreas, the blood sugar was 360 mgm. per cent, in the mother and 340 mgm. per cent, in the foetus, but at the beginning of the 7th week, when a similar experiment was tried, the values were respectively 274 mgm. per cent, and only 1 75 mgm. per cent. Although the protection of the embryonic insulin was not complete, yet the embryo was clearly defending itself against the diabetic state of the mother. Aron therefore divided the regulation of the foetal blood sugar into two periods, {a) an earlyone in which regulation depends solely on the function of the placenta, and {b) a later one in which it depends on the function of the insulinproducing islets of Langerhans as well. The problem of whether in the first of these two periods there is any control of the foetal blood sugar by the maternal insulin, Aron can hardly be said to have solved. Injections of insulin into the mother during the first period certainly lowered the foetal blood sugar, but not as much as the maternal, i.e. 20 to 25 per cent, of the normal instead of 50 per cent, or more of the normal. The conclusion seems justifiable that in period {a) the foetal blood sugar is kept by the placenta at a definite relation with the maternal blood sugar, and this may fall or rise as the latter falls or rises. Aron's conclusion was more complicated. He thought that the foetus, in normal conditions, received from the mother a utilisable form of glucose which it adapted to its own metabolism, and that, when the maternal pancreas was removed, this form of glucose was no longer produced, so that a foetal diabetic state would follow immediately upon a maternal diabetic state. It is difficult to see why this should have followed from his experiments. For further description of the work of Aron and his collaborators on the carbohydrate metabolism of the mammahan embryo, see Section
I5-3 Returning now to Fig. 289 it is obvious that the blood sugar falls in some cases and rises in others as development proceeds. As far as Aron's work went, it appeared to fall in the case of the pig, and to rise in those of the guinea-pig, rabbit, dog and cow. Further points were collected for the rabbit by Snyder & Hoskins but cannot be plotted, as the full data have not been published. These workers state that the foetal rabbit blood contains 33 mgm. per cent, at 22 days of gestation and 100 mgm. per cent, at 32 days — it thus rises and approaches the maternal level. Further work on foetal blood sugar levels is urgently required, especially in view of the interesting work of Scott, who in comparing the blood sugar of various mammals, found that it was highest in the smallest ones, i.e. those whose energy expenditure would be greatest (e.g. rabbit 107, guinea-pig 118, rat 138, mouse 245 mgm. per cent, among rodents).
The effect of glucose in the medium of embryonic cells in tissue culture was first studied by M. R. Lewis who found that its presence was essential and that lack of it led to vacuolation and death in cultures of connective tissue cells from 8-day chicks. Willmer, using intestinal cells from 11 -day chicks, confirmed this and found that the optimal concentration of glucose was rather lower than i per cent., though growth would sometimes proceed well in concentrations up to 2 per cent. When, however, Holmes & Watchorn came to make cultures of the embryonic rat kidney, they found that the optimal concentrations were much lower, varying around 0-2 per cent, or even a good deal less, and they suggested that the difference lay between avian and mammalian tissue. They recalled that the amount of glucose in the egg is at certain times quite large, and thought it likely that the chick's tissues might be exposed to greater concentrations of glucose in the egg than those of the rat in the uterus. As will be mentioned in the Section on protein metabolism, Holmes & Watchorn were able to show that glucose exerted a marked protein-sparing action on rat embryonic kidney tissue grown in vitro, as judged by the diminution in ammonia and urea-production. Krontovski & Bronstein, who also worked with embryonic tissues from the rat, were able to demonstrate by microchemical methods a disappearance of glucose from the medium in which the cells were growing!.
8-10. Carbohydrate Metabolism in Amphibian Development
We may now consider the movements of the carbohydrate fractions in eggs which have so far been left out of account. The reptiles have been very little investigated, but according to Tomita the 100 mgm. per cent, of free glucose present in the egg of the sea-turtle, Thalassochelys corticata, have disappeared entirely by the 30th day of development. Corresponding with this diminution there is a peak in lactic acid content (see p. 1053).
As regards amphibia, a histochemical study of the glycogen in the frog embryo and larva was made by Konopacki and by Konopacki & Konopacka. After fertilisation and the formation of the perivitelline space, the amount of glycogen thus estimated diminishes considerably, and apparently undergoes no further changes till the gastrula stage. At this time there is little to be seen; only a few cells of the blastula and gastrula have any in them. At the neurula stage, however, while the first organs are being formed, glycogen appears again, and rises in amount until it can be found in all the tissues all over the body. In later periods all organs do not behave in the same way, for in some of them the glycogen soon disappears again, while in others it steadily accumulates during larval life. Thus in the mesenchyme cells, the central nervous system and the eye-cups, its appearance at the neurula stage is only transient, and there is none there by the time that a length of 5 mm. is attained, but it persists much longer in all the epithelial cells, and definitely increases in amount in the skeletal and cardiac muscle. It will be observed that these findings are roughly in accord with those of Claude Bernard. Konopacki also studied the effect of the formation of the perivitelline space on the glycogen in the frog's egg, and found that it corresponded with a marked diminution of it — "le glycogene", he said, "disparait presque entierement", but the contents of the perivitelHne space gave a very strong reaction for glycogen, so that it would not appear to be lost from the system as a whole.
1 This was confirmed by Holmes & Watchorn and by Friedheim & Roukhelman. Acidification of the medium may occur owing to the lactic acid produced (Magaih) .
Thus the glycogen in the frog's egg at fertilisation found by Kolb; Luchsinger; Athanasiu; Bleibtreu; and Kato (see Table 46 and Appendix 11), is partly eliminated into the perivitelline space and almost wholly used up by the cells of the embryo before gastrulation. After that time more glycogen is formed and stored in the tissues, which after a preHminary general distribution, hand over most of it to the keeping of the liver and the muscles. The details were filled into this bare outline by the long memoir of Konopacki & Konopacka. Thus they showed that, after gastrulation, glycogen does not in general appear in endodermal cells, e.g. liver and pancreas, though the gill region is an exception. In the ectoderm it appears profusely, but does not persist there except in the epidermis, where it exists even after hatching. The organs of mesodermic origin take an intermediate position, for after gastrulation, glycogen is found to a considerable extent in them, but very soon disappears. The skeletal and cardiac muscles form an exception to this rule. No glycogen seems to be present at any time in the genital cells. During the hunger period after the yolk-sac has been all used up, if no food is provided for the larvae, glycogen disappears from all the places where it is stored. Conversely, if food is given, the glycogen stores increase throughout, and glycogen appears for the first time in the liver, the white matter of the central nervous system, the cells of the choroid plexus and the retina.
Konopacki & Konopacka insisted on the necessity of histochemical work as adjuvant to purely chemical investigations, and, as it is true that chemical analyses at present cannot distinguish between regions such as the three germ layers, for instance, they were quite right. But what they did not emphasise was the fact that histochemical methods are much more uncertain than purely chemical ones.
Faure-Fremiet & Dragoiu paid some attention to the glycogen in the developing frog's egg. Using the Bierry-Gruzevska method, they found 3-31 gm. percent, (wet weight) glycogen in the unfertilised egg, or 7-81 per cent, dry weight, and in absolute figures 0-135 mgm. per egg. At hatching the glycogen had diminished to 1-75 per cent, wet weight, or 0-079 n^gm- per ^gg, so that between fertilisation and hatching each egg had lost 0-056 mgm. of glycogen. It would thus appear that over the whole period there is a loss of glycogen, amounting to 41 per cent, of the amount originally there. Evidently the mechanisms at work in the frog's egg differ considerably as regards glycogen from those at work in the hen's. The fall in glycogen added to the fall in fat was found by Faure-Fremiet & Dragoiu not to account for the fall in dry weight and calorific value, so they postulated a fall in protein as well. At the end of the larval yolk-sac period, these workers could find only traces of glycogen. Over the whole period a dry weight loss of 17-3 per cent, was observed, of which 3-2 per cent, was contributed by glycogen, for Faure-Fremiet & Dragoiu did not envisage the possibility that the glycogen might have been transformed into some other substance.
This, however, was found to happen by Needham. Table 129 gives the results obtained, setting side by side with them the data of all the observers who have ascertained the loss in dry weight and the big gain in wet weight which the frog embryo has before hatching. It can thus be seen that, although Faure-Fremiet & Dragoiu found that the egg has only 58-5 per cent, of its glycogen left at the end of development, it has 92-8 per cent, of its total carbohydrate, and the conclusion must be that the glycogen has been transformed into some other kind of carbohydrate, not, as Faure-Fremiet & Dragoiu thought, that it has been combusted to provide energy for the embryo.
The balance sheet of the frog's development given in Table 129 shows that the average frog embryo gains about 2 1 per cent, in wet weight, and loses about 32 per cent, of its dry weight, while 41 per cent, of its glycogen disappears, but only 7 per cent, of its total carbohydrate.
o day 8 days Diff.
I 2 3
4-16 5-22 + 106
399 5-9
4-42 + 0-43 II +15-2
5-4 + 2-1 809 + 1-41
Average +4'62
Result (20-8 °/o)
Table 129. Balance sheet of the developing frog embryo (Rana temporaria).
Dry weight (mgm.)
o day 8 days DifT.
■9 63
3
38 33 £6
313
1-30
105 I-5I
Glycogen (mgm.)
o day 8 days
7 8
0135 0079
DiflF.
9 -0056
Total carbohydrate (mgm.)
o day 8 days Diff.
Investigator 13 Faur^-Fremiet &Dragoiu(i923)
29 — —
31 062
-0-65 (32-4 °/o)
— — — Faur^-Fremiet
& Vivier du Streel (1921)
— — — — — Bialascewicz
(1908)
— — — — — Bialascewicz &
Minc6vna(i92i)
— — — — — Bonnet & Barth^ lemy (1926)
— — — — — Williams (1900)
— — — — — Haensel (1908)
— — 00402 0-0373 —0029 Needham (1927)
— -0056 — — -0029 (41-5 °/o) (7-2°/ J
Method used
14
Bierry-Gru
Pfluger HagedomJensen
The amount of total carbohydrate found, using the HagedornJensen method, is rather less than the amount of glycogen found by the Bierry-Gruzevska method, though very Uttle less than the amount of glycogen found by the Pfliiger method. This contradiction does not invalidate the arguments given above, for the values in each case are probably relatively exact. It is also hkely that the total carbohydrate estimations are nearer to the absolute values than the glycogen ones, for the estimation methods for glycogen have never been very good, and are still under discussion. Thus Asher & Takahashi have criticised Pfliiger's method severely, and the newer method of Rona & van Eweyk, which unfortunately nobody has used on the frog embryo, gives much lower results than the older ones.
On the whole, therefore, the results of the chemical investigators agree very well with those of the histochemical ones. But it is wrong to conclude, when glycogen is seen to be disappearing histochemically, that it must be destined for combustion purposes. It may only be going to other forms of carbohydrate. This is one of the ways in which hurried conclusions from merely histochemical evidence may lead to confusion. It also shows again that glycogen cannot be considered as representative of the carbohydrate group.
One point which has resulted from these investigations of the carbohydrate metabolism of the frog embryo is that there is no glycogen in the liver until a very late date, so that Aron's views on the assumption of its function are strongly supported. Another point of interest is the probabiHty that the hatched tadpoles derive nourishment from the jellies around their eggs. We have already seen that there is some evidence that they do (p. 909). If this is the case, the stores of carbohydrate in the form of mucoprotein there may be an important source for the sugar of their bodies, especially as they can absorb substances through their skins. It is clear that the question of amphibian carbohydrate metabolism has so far only been touched on, and that there is much room for an extended investigation of it, including the determination of glycogen and free glucose by improved methods on each day before and after hatching as well as on parts of the animal.
8-11. Carbohydrate Metabolism of Invertebrate Eggs
A certain amount of work has been done on the carbohydrate metabolism of the eggs of the nematode worm Ascaris. Brammertz and Marcus, using purely histochemical methods, observed a diminution of the glycogen in the eggs following fertilisation. Then FaureFremiet estimated the glycogen before and after segmentation by the Pfliiger method, obtaining 1-75 per cent, dry weight before and 1-05 per cent, dry weight afterwards, i.e. a loss of 0-7 per cent, over the whole period. On the other hand, he obtained a figure of no less than 2 1 per cent, dry weight for the ripe ovary, most of which must have been in the eggs. Von Kemnitz reported histochemical observations which showed that, in the ovaries before the eggs were laid, they possessed large stores of glycogen. Immediately after fertilisation, however, Faure-Fremiet found only 4-67 per cent, of glycogen, though the chitin of the newly formed membranes accounted for an additional 9-23 per cent. The two added together did not equal the glycogen-content of the ripe ovarial eggs, from 7 to 9 per cent, being missing. "This quantity", said Faure-Fremiet, "has disappeared without leaving any traces, and as we know that the life of^Ascaris is essentially anaerobic, the lost glycogen not transformed into chitin cannot have been burnt. Weinland showed that in Ascaris glycogen can be transformed into lower fatty acids such as butyric and valerianic." Unfortunately, this is now quite discredited (Slater). After fertilisation, the disappearance of glycogen continues slowly, the percentage dropping from between 4 and 5 to rather less than 2, and during segmentation this again falls to about i. It will be remembered that in Section i-i2 (p. 328) reference was made to Faure-Fremiet's demonstration of the origin of the chitinous membrane of these eggs from their glycogen.
His work was repeated and for the most part confirmed by Szwejkovska. She found 4-85 gm. per cent, (of egg-contents) in the Ggg at fertilisation, 2-01 after the eUmination of the first polar body, and 0-756 after the elimination of the second. Of the 52 per cent, of the glycogen disappearing between the first two points, she found 47 per cent, as glucosamine in the chitinous envelope. Thus the chitin in grams per cent, rose — 1*665 per cent, at stage i, 3-39 per cent, at stage 2, and 3-507 per cent, at stage 3. There was no transformation of carbohydrate into fat, for the fatty acids also diminished in amount during the interval between fertihsation and the first cleavage:
Grams %
Volatile Non-volatile Total After fertilisation ... ... ... 0-455 0-526 0-981
After elimination of second polar body ... 0-343 0-361 0-704
There was no change in the nitrogen-content.
The silkworm Q.gg has also been the subject of researches on carbohydrate metaboHsm. The first estimations were those of Vaney & Conte, who noted a steady and gradual diminution of glycogen throughout embryonic Hfe, from 3-08 per cent, dry weight at the time of laying to 0-413 per cent, at the time of hatching. Their figures are shown in Fig. 290 beside the data of Pigorini and of Tichomirov, which correspond very well. There can be little doubt but that the glycogen-content of the whole silkworm ^gg falls markedly during the post-hibernation period, and it is probable that it rises slightly during hibernation, although it may then remain constant. Before hibernation the glycogen seems to fall a great deal also. Tichomirov pointed out that the formation of chitin in this insect (o-o to 0-21 per cent.) would probably account for some of the glycogen disappearing. To these researches we must add that of Kaneko, who noticed histochemically that at hatching no glycogen was present in the silkworm embryo or its egg, a finding quite in agreement with what has already been said.
Rudolfs, using the egg of the tent-caterpillar, Malacosoma americana, found a decline from 0-28 per cent, dry weight to 0-15 per cent, during development.
For echinoderm development we have only one complete piece of work. Ephrussi & Rapkine found for Strongylocentrotus lividus that the unfertilised eggs had 5*43 per cent, total carbohydrate, the gastrulae 5-46 per cent, and the plutei 3-4 per cent., i.e. at o, 12 and 40 hours respectively from fertilisation. The corresponding wet weight percentages were 1-36, 1-35 and 0-72. It was therefore clear that during the segmentation stages no carbohydrate disappeared. Utilisation amounting to 50 per cent., however, took place after the stage of gastrulation. The partition between combined and free glucose was also interesting, as follows :
Fig. 290.
Hours
Non-glycogen and free glucose Glycogen
% of the total carbohydrate
12
40
9^
i8-3 817
100
This state of affairs bears a resemblance to that found for the frog's egg by Needham, as mentioned above.
The glycolytic power of the sea-urchin's egg [Arbacia) has been studied by Perlzweig & Barron. Measuring the lactic acid formed under various conditions they obtained the following figures:
Unfertilised control
Unfertilised plus potassium cyanide
Fertilised control
2-8-cell stages control ...
Mgm. lactic acid per gram of eggprotein (experiments of varying duration, mostly 3 hours)
3-14 5-68 3-40 3-23
67-2
From this they concluded that lactic acid was formed normally by the developing sea-urchin's egg, and that if its oxidation was inhibited it accumulated as in all other cells. There was a hint of more rapid formation after fertilisation. It will be recalled that Meyerhof in 191 1 (see the Section on Respiration) did not succeed in demonstrating any glycogen or free glucose in unfertiHsed Strongylocentrotus eggs, but that Matthews in 1913 (see the Section on Constitution) had found a Hpoid in Arbacia eggs which contained sugar. In 1927 by improved methods Blanchard did demonstrate the presence of traces of glycogen in Arbacia eggs, though not the minutest amount of free glucose. Perlzweig & Barron determined to estimate the total carbohydrate in the eggs before fertilisation, and, using much the same technique as in Needham's studies on the frog, they found about 50 mgm. of glucose per gram of egg protein. It is probable, therefore, that the major part of the carbohydrate in Arbacia eggs is present as a mucoid.
As has already been indicated in the Section on Energetics, respiratory quotients closely approaching unity have frequently been obtained during the cleavage stages of echinoderm eggs. Barron has recently been able to fertilise Arbacia, Asterias and Nereis eggs in strictly anaerobic conditions, a finding which suggests carbohydrate metabolism (see p. 758), the cells piling up an oxygen debt. Moreover, direct measurements of glycolysis rate have been made by Ashbel on Paracentrotus eggs : revealing an augmentation of N.G.R. on fertilisation. One mgm. of tgg nitrogen produced 0-845 rngm- of carbon dioxide per hour from the glucose mixture before fertilisation and 2-32 afterwards. It would be still more interesting to compare this N.G.R. with that of later stages up to the free-swimming pluteus (cf Section 4-20).
Fig. 291.
8-12. Pentoses
This type of compound has a certain importance in relation to the development of the nucleic acids of the embryo, since Calvery has shown that, besides the ordinary hexose nucleic acid which occurs in animal tissues, the chick embryo possesses also a pentose nucleic acid not unhke that of plants. The only investigation of the pentosecontent of an egg is that of Mendel & Leavenworth, who estimated it in hen's and duck's eggs, using Tollens' method. Their figures are shown in Fig. 291, from which it is obvious that the pentose-content of the eggs rises, reflecting the increase in nuclear substance.
8-13. Lactic Acid
A good deal more is known about the metabolic behaviour of lactic acid in the hen's egg. It was first found there by Bonnanni in 19 14, who regarded it as a normal constituent and reported more in the white than in the yolk. He stated that the fresher the egg the more lactic acid it contained, that there was less in the winter than in the spring, and that by feeding sajodin to hens the lactic acid content of their eggs could be raised. Anno next found traces in the hen's egg-white before incubation, and a rise to the 4th day, but none in the yolk and no rise. Tomita estimated it each day during incubation, and his data are plotted in Fig. 292. An extremely sharp peak is to be seen in both yolk and white on the 5th day of incubation. Tomita drew attention to the coincident marked fall in free glucose, which he knew only through the work of Sato, and expressed the belief that the two phenomena were intimately associated. Perhaps the mechanism by which the lactic acid is transformed into some other compound is itself in course of development, and does not increase in activity as fast as the mechanism which produces lactic acid — this would lead to a temporary accumulation of the intermediate product.^ Unfortunately we do not yet know the real meaning of this accumulation. In order to penetrate a little farther into the working of these processes, Tomita injected both glucose and alanine into the hen's egg at the beginning of development. He found that the addition of glucose to the egg in this artificial way led to a 50-70 per cent, increase of lactic acid in the white, and to a definite increase in the yolk, though not to more than 15 per cent., and that in only one case. This apparent isolation of the yolk from the events going on in the white during the first week of incubation has been noticed before when we were considering the effect of injections of glucose on the free glucose of the yolk and white. It was interesting that the addition of 200 mgm. of glucose had no more eflfect in increasing the lactic acid production than the addition of 50 mgm., from which it may be inferred that the relationships considered cannot be simply governed by mass action (see Fig. 293). The lactic acid produced amounts at its maximum to about 46 mgm. per egg, and the amount of free glucose lost during the first 5 days is approximately 55 mgm. (see Fig. 267). When it is remembered that the estimate of the carbohydrate combusted by the 5th day is something very close to 10 mgm., the correspondence is remarkable, and leads to the inference that what is not burned can almost entirely be accounted for as lactic acid. After that we lose sight of it. The decrease in free glucose is more marked in the white than in the yolk (see Fig. 268), but the increase in lactic acid is more marked in the yolk than in the white. Comparative estimations of the lactic acid content of the white yolk and the yellow yolk would be of great interest.
Fig. 292.
Fig. 293.
- It is interesting in this connection that Neuberg, Kobel & Laser have shown the mechanism of lactic acid production in the chick embryo to be identical with that in other tissues. Acetone powders of 8-day embryos give with hexosephosphate good yields of methylglyoxal, showing the presence of active glycolase. The co-enzyme (co-zymase), which assists the ketonaldehydemutase in transforming methylglyoxal into lactic acid, has also been shown to exist abundantly in the embryo rat (Sym, Nilsson & v. Euler), mouse (Waterman), and pig (Kraut & Bumm).
Tomiba Effect of autolysis on egg lactic acid
Tomita's injection experiments were continued by Matsumoto several years later, who confirmed some of his normal figures and found that injected glycerol had not the least effect on the magnitude of the lactic acid content in either yolk or white. Tomita himself also studied the effect of autolysis on the lactic acid of the egg. As the diagram in Fig. 294 shows, no increase in the lactic acid of the white was to be seen even after 14 days' autolysis. The addition of glucose or alanine to this had no effect whatever, and no extra lactic acid was formed. The yolk, on the other hand, showed a marked rise in lactic acid when autolysed.^ As the second experiment demonstrates, it rose for about a week, but later it was found to fall, the lactic acid itself being destroyed. Addition of glucose to the yolk autolysate at the beginning of the experiment led to enormous rises in the lactic acid formed, e.g. from 30 to 300 or 400 mgm., while alanine gave no such increase, whatever its concentration. Tomita concluded from this that the enzyme which hydrolyses the free glucose into the lactic acid existed exclusively in the yolk. For the closely related work of Stepanek, see Section 14-6.
Tomita drew attention to the fact that the maximum figure for lactic acid obtained in normal autolysis was quite similar to the maximum observed during normal development.
Ido's experiments were planned rather differently. Taking hen's eggs after variable periods of normal development, he vaselined them so as to exclude air and returned them to the incubator for several weeks. Considerable amounts of lactic acid accumulated, but the correlation between lactic acid formed and glucose destroyed was only precise in the case of embryos at least as old as 5 days. In unincubated eggs thus treated only 2 7 per cent, of the glucose disappearing could be accounted for by the lactic acid formed. These experiments are of interest in connection with Byerly's findings (see p. 607).
^ Confirmed subsequently by Needham & Stephenson.
Fig. 294.
The hen's egg is not the only type which has been examined with respect to lactic acid. Yoshikawa in 191 3 found 3 mgm. per cent, in the white and 1 2 mgm. per cent, in the yolk of the fresh egg of the marine turtle Thalassochelys corticata, and the subject was later pursued by Sendju. It turned out that just as Tomita had found a peak of lactic acid in the development of the bird so Sendju found one in that of the chelonian reptile. Beginning at 8 mgm. per cent, in the fresh egg, the lactic acid rose to 41 per cent, on the 15th day, falling to 1 5 per cent, on the 45th day. A high value for the newly hatched tortoises may perhaps have been due to the muscular effort involved. The general trend of the figures affords another indication of the similarity between the general metabolic picture of the various sauropsida in pre-natal life.
8-14. Fructose
Attention must finally be given to the fructose question which is the most enigmatic aspect of embryonic carbohydrate metabolism. It begins with Claude Bernard, who in 1855 noted that the sugar of the human amniotic liquid was laevorotatory, but that this condition was no longer present at term. In 1904 Giirber & Grunbaum observed that 40 per cent, of the reducing carbohydrate of the amniotic and allantoic fluids of the horse and pig was fructose. This was confirmed and much extended by the classical researches of Paton, Watson & Kerr in 1907, who reported the presence of fructose in the amniotic and allantoic fluids of the sheep, cow, and probably the dog. The blood of the sheep embryo contained in one experiment 420 mgm. per cent, of fructose, but it was not demonstrable in the liver. Then in 1922 Takata found that fructose was the only carbohydrate present in the amniotic fluid of the whale Balanoptera, and not long afterwards Orr noted that human and goat foetal blood give the Selivanov reaction, and have an abnormally low rotation. In human foetal blood the fructose/glucose ratio seems to be 1/2 and the former sugar does not completely disappear at birth. There may also be a fructosuria of pregnancy (van Creveld & Ladenius). Are the rare cases of fructosuria in adults cases of arrested development, just as icterus neonatorum seems to be a continuation of a normal pre-natal condition?
Cite this page: Hill, M.A. (2024, April 27) Embryology Book - Chemical embryology 2-8 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-8_(1900)
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