Book - Chemical embryology 2-10 (1900)
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Needham J. Chemical Embryology Vol. 2. (1900)
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Chemical Embryology - Volume Two
Section 10 The Metabolism of Nuclein and the Nitrogenous Extractives
10-1. Nuclein Metabolism of the Chick Embryo
It has already been recounted in Section i (see p. 336) that many investigators, following Kossel, have not been able to isolate any purine bases from the unincubated hen's egg. At the present time it is generally agreed that not more than 2 per cent, of the total nitrogen at the beginning of development is in the form of purine bases. All investigators have agreed, moreover, that by the end of development purine bases are present in the embryo in considerable quantity, and there is no doubt that, during the embryonic growth of the chick, purine bases are synthesised. The only dissentient voice which breaks this unanimity is that of Mesernitzki, who maintained in 1903 that as much purine (estimated as xanthine) was present in the egg at the beginning as in the embryo at the end, but we may disregard it, for Fridericia adversely criticised his technique, and repeated his experiment with contrary results.
Fig- 343
Sendju's paper contains figures for the purine nitrogen in white, yolk and embryo, and is probably the best one with which to introduce the subject. As is evident from Fig. 343, there is never more than a minimal quantity of the bases in the egg-white, but in the yolk, on the contrary, there is a good deal, and on the 14th day it contains as much as the embryo. The graph accordingly resembles that for glycogen, for it is of the type in which the total amount of the substance increases, while the amount of the substance outside the embryo first increases and then decreases. Probably the reason why the yolk has so much is that the yolk-sac and its vessels were included in the analysis, and consequently the purine bases from the nucleoprotein of their cells were included. Mendel & Leavenworth have also estimated the purines in the avian egg, and their figures, which appear in Fig. 343, agree with Sendju's as demonstrating a vigorous synthesis, although in absolute value they much exceed his, by 22-0 to 7'0 on the last day of development. This is due to the fact that Mendel & Leavenworth probably included the uric acid present.
Mendel & Leavenworth separated a number of the individual bases by the Kruger-Schmid process, with the results shown in Fig. 344. Kossel's original figures are placed beside them for comparison. The concentration of the bases in the embryonic tissue was, it seemed, about the same as in adult tissue, thus:
Grams % wet weight
Mendel 8c Leavenworth © Guanine "j
e Adenine >WhoU
® HypoxanbhineJ
Kosse O Guanine 1
O HypoxanbhineJ
Fig. 344.
Hypo
Guanine
Adenine
xanthine
Xanthine
Cow muscle : embryo
0-044
—
0-038
0-012
Kossel, 1884
adult
0-005
—
0-053
0-012
Pig muscle :
embryo (100 mm.)
0-093
0-065
0-029
Mendel' &
adult
0-027
0-059
—
Leavenworth 1908
Wells & Corper remarked in 1909 that foetal tissues seemed to contain much more guanine than adenine, since on autolvsis they gave twice as much xanthine as hypoxanthine. No more recent investigations have been made exactly along these lines, but Calvery has reported the presence of pentose nucleic acid with its pyrimidine, uracil (the so-called "yeast nucleic acid"), in the i8th-day chick embryo, as well as the ordinary hexose nucleic acid similar to that first isolated from the thymus. The chick embryo thus possesses the power of synthesising both kinds of nucleic acid. The older observations of Mendel & Leavenworth therefore regain their importance, for, using Tollens' method, they found an increase in the pentose content of the incubated egg from zero to as much as 40 mgm, (see Fig. 345).
The first systematic investigation of the synthesis of nuclein bases by the embryo was made by Fridericia, who estimated them in the chick embryo from the 8th day onwards, using the Brugsch-Schittenhelm technique. He fully confirmed the earlier statements that not more than traces of purine bases were present in the unincubated egg, e.g. 0-25 mgm. purine nitrogen per egg. In order to determine whether the presence of any purine bases tightly enough combined to resist acid hydrolysis had been overlooked, Fridericia allowed fresh eggs to autolyse, but did not obtain in this way any more purine nitrogen than by the previous method. The small amount of nucleoprotein in the fresh egg is doubtless to be accounted for by the blastoderm itself. His figures for increase of purine nitrogen in the embryo are given in Fig. 346. Fridericia compared together in one and the same graph the absolute amounts of dry weight, total nitrogen and purine nitrogen. From the 8th to the i ith days the rate of increase was much the same, but after that the dry weight and total nitrogen curves drew away, and the purine nitrogen one was left slowly increasing below them. This obviously indicated that the purine nitrogen would decrease markedly in per cent, of the total nitrogen, but Fridericia did not pursue the subject further. Its importance will shortly appear.
More recently, the question of nucleoprotein formation has been
Fig. 345
lembranes (Fridericia)
" (LeBrebon^Schaeffer)/
(Targonski) " ( Sagara)
examined by LeBreton & Schaeffer, who made a critical study of the possible methods, and eventually used their own modifications of the Kruger-Schittenhelm method. Their programme involved the estimation of the total nitrogen and the purine nitrogen at "I" Purine-nitrogen different stages in several series of embryos. Their data on the total nitrogen of the chick have already been given in Table 136 of Section 9-3, and their findings with regard to its increase in purine bases are shown in Fig, 346.
Fig. 346.
Targonski calculated the relation between the "anabolic" and the "catabolic" purines, in order to see how much purine nitrogen out of the total made
each day was excreted into the allantoic liquid, and how much was stored up in the cells. He used his own figures for both sets of data, as follows :
Milligrams uric
Milligrams
acid nitrogen
purine nitrogen
per gram wet
per gram wet
weight embryo in
weight embryo in Anabolic purine/
Day
allantoic liquid
embryonic body catabolic purine
• 10
0-28
0-65 2-3
12
0-29
0-69 2*4 0-72 1-8
\t
0-39 078
0-77 i-o
18
0-44
0-85 i-g
The ratio was, he concluded rather curiously, a constant, and meant that about 30 per cent, of the purine nitrogen formed on any one day is catabolic and 60 per cent, anabolic. In view of the considerable differences which have already been noted between the absolute values of different workers for uric acid production, it is difficult to decide whether these relations have any significance. Moreover, wet weight is obviously less satisfactory than dry weight for such a comparison. I therefore recalculated what may be called Targonski 's ratio, using Murray's figures for dry weight, those of Fridericia and of LeBreton & Schaeffer for purine nitrogen, and my own for uric acid production. The justification for this is that all these investigators used White Leghorn embryos. As Fig. 347 shows^ the ratio, far from being, as Targonski thought, a constant, changes very markedly. Attention may first be concentrated on the curve obtained from LeBreton & Schaeffer's data, i.e. the curve for embryo alone, the membranes not being included. It begins at a very high value but immediately descends to reach a minimum on the 1 1 th day, or, in other words, a condition when the excreted purine — the uric acid — somewhat exceeds the architecturally utilised purine. This minimum precisely corresponds, as is shown by the vertical line on the graph, with the point of maximum intensity of uric acid production, which has been already described, and which is pictured in Fig. 323. Later, Targonski's ratio rises again, and then falls slightly, although the uric acid excretion always exceeds the layingup of purines in the cells. If now the Fridericia curve is considered, it will be seen that the ratio does not dip below the unity line till the i8th day, which shows that, if the purines in the membranes are taken into consideration, then the anabolic purines rather exceed the cataboHc purine during most of development. The first trough is present, although shifted some 2 or 3 days forward, but the time of the subsequent peak is little changed. In short, Targonski's ratio varies during development with the intensity of protein metabolism, as we should expect from the fact that the main endproduct of protein metabolism in the bird is a purine ring.
10-2. The Nucleoplasmatic Ratio
This expression was originally introduced by R. Hertwig, who in 1 903 suggested that there was a definite relation between the mass of the nucleus and the mass of the cytoplasm. "The diminution of the nuclear mass seems, as Boveri has shown, to bring with it a diminution in cell size; the augmentation of the nuclear mass, according to Gerassimov, leads to augmentation of cell-size." The relation between the two was rapidly taken up by morphologists,
Fig. 347
who evolved before long more or less quantitative methods of measuring it. As the basic nature of the work was always microscopical, these methods were restricted to the measurement of areas and surfaces. The school of Godlevski, which was pre-eminent in this field, used the method of drawing the cell and nucleus with a camera lucida, and then cutting out the drawing, pasting it on cardboard, and weighing the cardboard. Details of this method may be found in Godlevski's paper of 1 92 1. It could not commend ^^ itself to anyone with a physico- ■chemical turn of mind, for it J depended on the assumption i that the surface area as seen f microscopically bore a uniform I relation to the mass, which, in s view of the changing compo- "^ sition of nucleus and cytoplasm, i may not be the case at all. < Nevertheless, as a rough approximation the method was useful. Godlevski found great changes in the N.P.R. during maturation and segmentation in echinoderm eggs. Each stage has a characteristic N.P.R. :
Table 164.
500 Number of nuclei
ibryo
Fig. 348.
'Total volume of
nuclear material
Nucleus
Cytoplasm
cubic IX
volume
volume
Unfertilised egg ..
650
550
Fertilised egg
1300
275
2-cell stage
1382
275
4-cell stage
2084
177
32-cell stage
i9>938
64-cell stage
30,262
12
Blastula ...
30,716
6
Thus as the nuclei become more numerous and individually smaller, the total mass of nuclear matter in the whole egg increases enormously. Other good series are those given by Enriques for Aplysia (Fig. 348) , by Bury and by Erdmann. Similar work was done by Conklin. But to summarise all the researches which have been made on the morphological N.P.R. would serve no purpose, for they have already been reviewed in the monographs of Morgan, and of Faure-Fremiet.
But a definite step forward was made by LeBreton & Schaeffer when they translated the older conception of N.P.R. into physico-chemical terms, and defined it as the expression:
Purine nitrogen x lOO Total nitrogen — Purine nitrogen'
Two separate ideas seem to have been in the minds of the cytologists who dealt with N.P.R., firstly, that they were measuring simply cell and nuclear volume, and, secondly, that they were measuring somehow the amount of nuclear substance, or even nucleoprotein, present in the cell. LeBreton & Schaeffer's advance did not affect the first of these ideas, for microscopical measurement of N.P.R. still remains valuable cytologically, but it did supersede the second notion with a real hope of exactitude. It had always been obvious that nucleoprotein might be dispersed to some extent in the cytoplasm, and might not be all in the nucleus.^
One paper existed already in the literature in which definite information had been given about the "chemical" N.P.R., although its author did not perceive the full significance of his experiments. This was the memoir of Masing who estimated the nucleoprotein phosphorus in rabbit embryos according to the method of Plimmer & Scott, and obtained the following figures, which it is impossible to plot, in view of the imperfect characterisation of the material.
Table 165.
Nucleoprotein phosphorus
in milligrams per lOo mgm.
Whole rabbit embryos :
of total nitrogen
15 mm.
5-8
21 gm. 4th week
5-1
22'5 gm. 4th week
4-85
28 gm.
4-2
36 gm. 2 days before birth ...
3-7
43 gm. Birth
3'45
After birth
3'3
Rabbit hvers :
Beginning of 4th week
t^
Later stage
I day before birth
5-1
Birth
4-85
From this it was evident that the nucleoprotein as related to the total protein of the body or organ diminished during development. The result of LeBreton & Schaeffer for the chick are shown in Fig. 348, from which it again appears that the chemical N.P.R. falls during development. Since Fridericia collected the same facts, it is possible to calculate a chemical N.P.R, from his figures, and Fig. 349 shows that a notable fall occurs in his data too.
- In recent times it has been convincingly shown that the greater part of the purine of adult muscle is present as (probably cytoplasmic) nucleotide and not as nucleoprotein. Studies on the N.P.R. must in future take this into account.
LeBreton & Schaeffer also investigated the N.P.R. of the pig and the mouse (Fig. 351). They both descend in the same way as that for the chick. LeBreton & Schaeffer laid some stress, possibly
Fig- 349
Fig. 350.
not quite justifiably, on the absolute value of the N.P.R. at the earliest stage, interpreting the fact that it fell from 1 2 in the case of the chick, and only from 7 in the case of the pig, to mean that the former was the seat of more intense transformations than the latter. In other words, they introduced a conception of potential, and suggested that perhaps differences in gestation time and life-span might be due to such differences, the chick, as it were, being suspended at a higher initial level than the pig, and correspondingly falling more rapidly to equilibrium. The forms of the curves led them to suggest that probably the most rapid fall would always be associated with the highest initial values: thus the chick's N.P.R. descends from ii to 4 in just over 10 days, but the pig's N.P.R. takes 70 days to descend from 8 to 4. "La hauteur du potentiel reaHse au moment de la 'mise
, ,1 11 , 1 , lOr- LeBreton 8c,Schaeffer N.P.R
en charge des cellules de la ^ blastula ne determinerait-elle pas le regime de decharge ulterieurement realise pendant le developpement?" said LeBreton & Schaeffer. "La decharge se ferait d'autant plus vite que lavaleurinitialeduN.P.R.serait plus elevee." It is as yet too early to say what fruit, if any, this interesting suggestion will bear, but it gains interest from Rubner's well-known table, which shows that the intensity of chemical transformations is greater the smaller the animal and the shorter its gestation time. This subject has already been discussed in some detail in Section 2.
Fig- 351
Table 166.
Nitrogen in grams fixed
Number of days
daily by
Duration of required to
100 gm.
foetal life
double the
nitrogen during
Initial height
in days
birth weight
this period
of N.P.R.
Man
280
180
0-36
Horse
340
60
—
Cow
285
47
i'4
—
Sheep ...
150
13
4-4
—
Pig
118
15
4*7
7-5
Guinea-pig
67
Dog
63
8
7-4
—
Cat
56
i
7-3
—
Rabbit ...
28
II-O
—
Mouse
21
—
—
lO-O
Chick
II
—
—
"•5
LeBreton & Schaeffer also pointed out the resemblance between the fall of N.P.R. and metabolic rate (compare Fig. 349 with Fig. 143). From the figures now at their disposal LeBreton & Schaeffer were able to calculate the intensity of manufacture of purine nitrogen by the embryo. Smoothing their data for daily increments, they expressed these in terms of 100 mgm. of purine nitrogen, and obtained the curve shown in Fig. 352. Fridericia's data were also treated in this way, but with quite different results, for, whereas the figures of LeBreton & Schaeffer give a markedly peaked curve at 12 days of development, those of Fridericia give an amorphous assemblage of points. LeBreton & Schaeffer pointed out that his figures included the purines of the membranes, and concluded that when their masking effect was removed by omitting them from the estimations — as they themselves had done — then a clear maximum of intensity of production of purine bases revealed itself In order to have this important fact in a comparable form with other intensity curves, I recalculated the data in relation to dry weight (figures of Murray), with the result (shown in Fig. 353) that now both Fridericia's and LeBreton & Schaeffer's curves give a clear-cut peak about the 12th day. No one has had any explanation to offer of this peak in purine nitrogen production, but LeBreton & Schaeffer themselves hinted that it might be a curve of the same nature as the increment curve of an autocatalysed monomolecular reaction. They stated that their data for the pig and mouse embryo led to similar peaks.
Fig. 352.
Fig. 353
As for the fall in N.P.R. itself, they mentioned the obvious relation between it and the fall in percentage growth-rate (see Section 2). The higher the growth-rate, the higher the N.P.R. , i.e. the more purine nitrogen present per cent, of total nitrogen — but the connection between the two remains enigmatic. Defining further their notion, LeBreton & Schaeffer went on to say that with senescence the total nitrogen "grows" more rapidly than the purine nitrogen. They considered, further, that the N.P.R. would probably be found to rise from fertilisation to the blastula stage, and then to descend, but so far no determinations have been made which support or throw doubt on this view. LeBreton & Schaeffer themselves found the ripe eggs of the mackerel [Scomber scombrus) to have an N.P.R. of 1-5 and the adult liver cells one of 4-4, so that, if the latter had decreased from a much higher value, the former must have increased to it. More work along these lines would be very desirable.
10-3. Nuclein Synthesis in Developing Eggs
It has already been said that purine bases are undoubtedly synthesised by the developing chick embryo. The same holds for the embryo of the silkworm, if we may accept the early data of Tichomirov, who isolated 0-02 per cent, wet weight of total purine bases from the hibernating eggs, but over ten times as much, 0-23 per cent., from the fully developed embryos, of which about half was xanthine, and the rest hypoxanthine, guanine and adenine.
Similar results were obtained for the egg of the cod, Gadus morrhua, by Levene, although his methods were of doubtful validity.
Total purine
bases % wet weight
Unfertilised eggs ...
I day's development
II days
20 days
0-I2 2-l6 2-14
3-75
Perhaps the increasing basic nitrogen found by Gortner in his trout and salamander eggs may indicate an increase in purine nitrogen. In the latter case especially there was a gain of nitrogen in the etherinsoluble-but-alcohol-soluble fraction of 4-1 mgm. nitrogen, or 0-7 per cent, of the total nitrogen, which he ascribed to the synthesis of purine and pyrimidine bases.
The only investigator who actually isolated purine bases from echinoderm eggs was Masing. In the unfertilised eggs of Arbacia pustulosa he obtained i2-6 mgm. purine nitrogen from 2-96 gm. of dry substance, or 0-425 mgm. per cent., and from those in the morula stage 1-721 gm. gave him 7-56 mgm. nitrogen in the purine silver precipitate, or 0-439 mgm- per cent. In all cases 100 mgm. of total nitrogen contained 4-6 mgm. of purine nitrogen. In just the same way there was no change in the amount of nucleoprotein phosphorus up to the morula stage (see p. 1245). Masing's results were afterwards criticised by Robertson & Wasteneys on the ground that his material must have contained excess of spermatozoa, which are, of course, very rich in nucleoprotein, but this was not justifiable as Masing's unfertilised eggs gave as much purine nitrogen as later stages. Subsequent work by Needham & Needham on various invertebrate eggs strongly supported Masing, for the nucleoprotein phosphorus was found to be constant during development.
Nucleoprotein mgm. per gm.
phosphorus dry weight
Before development
2-22
s 1-95
After development
1-59 1-41
Sand-dollar (Dendraster excentricus) Starfish {Patiria miniata) Sand-crab (Emerita analoga) Brine-shrimp {Artemia salina)
These facts are interesting in view of the great increase known to take place in the total quantity of microscopically visible nuclear matter during echinoderm development (see the figures of Godlevski given on p. 11 5 1 ) . All the early workers believed that nuclein synthesis occurred in echinoderm eggs, partly because, like Robertson, they were convinced that the choline of lecithin was the causative agent in cell-division, and the lecithin phosphorus must therefore be used in other ways, and partly because, like Loeb, they were impressed by the behaviour of the nucleoplasmatic ratio and identified nuclear chromatin with the oxidations of the cell on the one hand and with the supposedly autocatalytic character of growth on the other. The flaw in these arguments was the identification of nuclear material as seen through the microscope with nucleoprotein as measured chemically. With regard to the marine invertebrate eggs so far studied we must on the contrary picture an organisation of preformed nuclein into the chromatin of the nuclei rather than a chemical synthesis of it from other raw materials.^
1 An apparent contradiction exists here. The histochemical method of Feulgen and Rossenbeck, which reveals the presence of nuclein, depends on a reaction of the aldehyde group of the hexose constituent with fuchsin sulphonic acid. J. Brachet has shown that
1158 THE METABOLISM OF NUCLEIN AND [pt. iii
Yet if this is what happens in the simplest alecithic eggs, there is abundant evidence that it is not so in the most compHcated ones. The preceding pages have demonstrated that the chick makes most of its nucleoprotein itself. We know practically nothing about this aspect of reptilian development, but the silkworm, as has been mentioned above, resembles the chick in nuclein synthesis. When, however, we pass to aquatic vertebrate eggs we find that the conditions are reversed, not because we know what occurs during development, but because notable quantities of nucleoprotein are found in the undeveloped eggs, in agreement with those of aquatic invertebrates and in contrast to terrestrial animals. Ichthulin itself, as has been described in Section 1-13 almost certainly contains no purine bases, but when the eggs have been worked up as a whole, investigators have found them (Levene & Mandel and Mandel & Levene on cod, Tschnernorutzki and Steudel & Takahashi on herring, and Konig & Grossfeld on herring, carp, cod, pike, and sturgeon eggs). Henze isolated over i per cent, of pentose from dried octopus eggs. Finally it is likely that nematode eggs contain large stores of nucleoprotein for Faure-Fremiet found a good deal of the phosphorus to be combined in that way. We may summarise these facts in the following table :
Nuclein phosphorus
In per cent
.of the
% of the
total phosphorus
final
.
amount
In the un
In the
present
developed
finished
at the
egg
embryo
beginning
Aquatic.
Sand-dollar (Needham & Needham)
32-0
26-7
1000
Starfish
156
25-4
6i-o
Gephyreanworm,, „
15-8
54-1
34-6
Sand-crab „ „
IO-8
139
77-9
Brine-shrimp „ „
37-9
27-4
1000
Sea-urchin (Masing)
lOO-O
Frog (Plimmer & Kaya)
^6
268
28-5
Terrestrial.
Silkworm (Tichomirov)
By purine
bases
95
Hen (Plimmer & Scott)
1-9
27-2
7-0
there is nothing like enough nucleic acid (as judged in this way) in the fertilised eggs of invertebrates to provide for the increase in nuclear material during development. If the nucleoprotein of the egg, however, contained pentose and not hexose, it would not give the Feulgen-Rossenbeck reaction and this, Brachet suggests, is the explanation of the discrepancy. Such an explanation is particularly plausible in view of the work of Calvery (seep. 1 147).
Without formulating any definite generalisation it may be said that this association between nucleoprotein synthesis and terrestrial life is probably no coincidence. In Section 9-15 it was shown that embryos which develop in terrestrial, i.e. highly cleidoic, eggs excrete their waste nitrogen mainly in the form of uric acid, and this fact was advanced as an explanation for the existence of uricotelic metabolism in the birds, reptiles, and insects. Now if the uricotelic mechanism is essentially due to the presence of the enzyme uricoligase, which will synthesise the purine ring from lactic acid or some other three-carbon chain and ammonium carbonate or urea, it is interesting to find that wherever the uricotelic power exists, there also in the egg a notable synthesis of purine bases for chromatin exists, and where ammonia or urea are the end products of protein catabolism, there the egg is provided with sufficient or nearly sufficient purine bases at the beginning with which to construct its embryo. It is as if the synthesis of the purine ring were relatively difficult, and just as in the mammals there is a return to urea excretion after the uricotelic metabolism of the reptiles, so in the transition from amphibia to sauropsida, supplies of nuclein being no longer necessary in the egg there is a retention of this important material by the parent organism. It will be very interesting to see if future work supports the provisional rule that only uricotelic embryos synthesise nuclein.
As for the means by which this is accomplished nothing is known.
Russo regarded his results as supporting the theory of ar ginine and histidine as precursors, but, as we have seen, Phmmer & Lowndes' results are in flat opposition to this view. Clemen ti has suggested that the protamines and his tones, with their extremely high arginine-content, are in reality the intermediate products between the amino acids of the yolk and the purine bases of the cellular nucleoproteins, but this is admittedly only a working hypothesis, and the subject is still one of the most obscure corners of the whole field.
Fig- 354
10-4. Creatinine, Creatine and Guanidine
The presence of creatine and creatinine in the unincubated avian egg was at one time a matter for dispute. In 191 1 Salkovski and Kojo independently reported the existence of traces of creatinine in the fresh egg, akhough four years before Mellanby had completely failed to find any there. However, there has always been agreement that organised embryonic tissues contain creatine and creatinine. Krukenberg in 1880 detected the presence of the latter substance in the muscle tissue of foetal calves at various ages. Later, Mendel & Leavenworth isolated 0-03 per cent, creatine from the muscles of a 265 mm. foetal pig — a low figure, the adult value being 0-45 per cent. Mellanby himself estimated the creatinine in the embryonic chick from the 14th day onwards, and some years later I obtained a series of figures parallel with his, but rather lower. These are shown in Fig. 354. There was little doubt but that the substance was present from the very beginning of development, and in 1924 Tiegs was able to demonstrate it by a colour test in the heart of the 4th-day embryo and in the general body tissue on the 5th day. Still later Fiske & Boyden reported a soUtary value for the 8th day of development, also shown in Fig. 354,
Fig. 356.
The subject was gone into in more detail by Sendju, who estimated the creatine and creatinine in yolk, white and embryo. As Fig. 355 shows, the preformed creatinine never exceeds i mg. in the embryo, while the creatine rises to about 8 mg. The amounts contained in the yolk and white are at all times negligible, and it is evident that a \dgorous synthesis must be proceeding.
The allantoic liquid, as has already been stated, contains creatine and creatinine. The concentrations of these substances rise steadily during development, as Fig. 356, plotted from the data of Fiske & Boyden and of Kamei, demonstrates.
As regards the origin of creatine and creatinine in the egg, nothing is known. The only attempt which has been made to find out is that of Burns, who in 1916 estimated the total guanidine in the hen's ^gg throughout incubation. The guanidine molecule is very resistant to oxidation, and Kutscher found that if a soluble protein is treated repeatedly with boiUng calcium permanganate, a mixture of oxidation products is obtained from which much can be driven off as aldehydes, ammonia, carbon dioxide, etc., and from which the guanidine can be precipitated with picric acid. This was the method employed by Burns, who operated on whole eggs including everything except the shell. Fig. 357, taken from his paper, shows the grams of guanidine picrate found in per cent, of the egg-contents. There is clearly a steady increase in total guanidine picrate from 0*3 to 2-8 per cent, at the 12th day, after which there is a falling
Fig. 357
74-2
Il62
THE METABOLISM OF NUCLEIN
[PT. Ill
off until the end of development. The formation of creatine cannot account for the decline in the curve during the latter part of development, for creatine and its anhydride were readily oxidised by the process used, and guanidine coming from them would be included in the figures for total guanidine. The total guanidine would have been expected to remain constant. Burns thought there were two possibilities, ( i ) that the configuration of the protein molecule of the chick might be more resistant to permanganate oxidation than the protein molecule of the yolk and white, or (2) that the guanidine precursor of creatine in the protein molecule may be partially destroyed by the oxidation process, just as enzyme action may sometimes split a protein through the guanidine group. Neither of these explanations is very satisfactory, for they both tend to throw doubt on the accuracy of the method as a test of the total guanidine present, and so to remove significance from the curve. A third possibility might be that after the mid-point of development the missing guanidine may be transformed into resistant rings such as pyrimidine or iminazol compounds. It is to be hoped that further work will be done on these obscure subjects.
The best determinations of creatinine in developing organs of a mammalian foetus are those of Beker given in Fig. 358. The increase seems to be exceedingly regular.
Hunter has found 546 mgm. per cent, creatine in the muscle tissue of the ovoviviparous dogfish, Squalus sucklii, at a time when the muscles of its foetuses (7 cm. long) gave 460 mgm. per cent. Probably, therefore the elasmobranch foetuses accumulate the substance in much the same way as mammalian ones.
Fig. 358.
Cite this page: Hill, M.A. (2024, April 19) Embryology Book - Chemical embryology 2-10 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-10_(1900)
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