Book - Chemical embryology 2-6 (1900)
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Needham J. Chemical Embryology Vol. 2. (1900)
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Chemical Embryology - Volume Two
Section 6 General Metabolism Of The Embryo
6-1. The pH of Aquatic Eggs
It will be convenient to take first the work on the single egg-cell, which has mostly been done on the eggs of marine invertebrates, and then to go on to the experiments which have dealt with the tissues and surrounding substances of such an embryo as that of the chick. Seven principal methods have been used in studying intracellular hydrogen ion concentration: (i) vital staining, (2) microinjection of dissolved indicators, (3) micro-injection of solid indicators, (4) " micro-ecrasement " or microcompression, (5) electrometric measurement by means of micro-electrodes actually in the cell,
(6) electrometric measurement upon thawing crushed masses of eggs,
(7) contact of indicators with crushed tissues. Each of these methods suffers from certain disadvantages. A critical comparison of them has been made by Reiss in his monograph on the subject, but his views, which are substantially those of the Strasburg school, have not been generally accepted. Vital staining is probably the least valuable of all the methods, for the dye may not penetrate and show a colour in the cell-interior until the cell has become completely abnormal^. Yet this, of course, was the general technique employed by the earlier workers, who would have preferred no doubt to use a pigment already naturally in the cells, as had been done in other cases, if eggs containing a natural indicator could have been found. The first observations were made by Schucking in 1903, who on inadequate basis regarded the granules in echinoderm eggs as alkaline and the protoplasm as acid. Then Loeb in 1906 stained sea-urchin's eggs with neutral red, expecting on theoretical grounds to observe a trend towards the acid side after fertilisation. This he failed to do, the dye absorbed by the eggs of Strongylocentrotus purpuratus from i/ioo molecular solution being quite red both before and after fertilisation. He noticed that after remaining 20 minutes in ordinary sea water, the unfertilised eggs became colourless, but the fertilised ones retained their colour. Parthenogenetic eggs gave the same effect. He also noted that the more development proceeded the deeper the eggs stained, and the more difficult it was to wash the stain out. He concluded that the internal pH was on the acid side of neutrality, but not much below 6-o. Moreover Warburg; Harvey; and Herlant all found by staining with neutral red that the contents of the echinoderm egg was more acid than sea water. Still earlier work by Keeble & Gamble, who had succeeded in staining Hippolyte and Mysis eggs with litmus, had indicated an internal pYL of between 4 and 3, but it is probable that the eggs so stained were dead.
1 And even if it does, it may undergo chemical change at the cell-surface.
Other work involving the staining method was even less illuminating. Faure-Fremiet, exposing the eggs of the polychaete worm, Sabellaria alveolata, to the action of such dyes as Nile blue hydrochloride, Nile blue sulphate and brilliant cresyl blue, concluded that a pYi of nearly 13 was reached at fertilisation. This extraordinary result was clearly due to the use of dyes which even under the best conditions are not good pH indicators, and which could not be trusted in a medium containing fatty substances, for which they have a special affinity. Lewis, again, growing embryonic fibroblasts on nutrient media to which indicators had been added, could not get any of them to stain until death occurred, whereupon they indicated a cytolysis />H of below 5-0.
The study of intracellular /?H by vital staining received a considerable impetus in 1925 from the work of Rous and his collaborators, who employed the method largely on the whole organism in mammals. Using his method, Harde & Henri found the following values for the mouse:
Uterine wall ... ... 6-0-6-2
Placenta ... ... ... 7-2-7-4
Embryonic skin ... ... 5"6-5*8
Maternal skin ... ... 7-4-8-6
Embryonic blood ... ... 5-8
This last value was in good agreement with another obtained in a similar way by Mendeleef on guinea-pig's blood, which she found to be at pa 5*8, although the maternal blood was />H 7-4. The tissue fluid of the embryo (how prepared?) she found to be at/?H 6-o.
It is, of course, doubtful as to what exactly is meant by intracellular />H or the internal j&H of the egg. There m.ust without doubt be numerous phases, perhaps of ^videly differing />H, and all that we measure directly by the various methods is the "j^H globale" or overall pYi of the cell. Lucke drew attention to the fact that, even when a cell seems uniformly stained, the dye may yet be in close association with minute granules or globules, and may be registering their internal pH. instead of that of the continuous phase in which they are. Lucke centrifuged the eggs of Arbacia and Cumingia, so that four layers appeared in them, a lipoidal one at the top, next a clear empty homogeneous protoplasmic layer, then a layer of small granules and at the bottom a layer of pigment granules. Such zoned eggs placed in 1/40,000 neutral red or brilliant cresyl blue solutions, took up the dye, but only as regards the granules. The clear layer and the lipoidal layer remained quite unaffected, unless they were exposed to the dye so long that they became granular, but that was an irreversible change leading to death. The centrifuged eggs were normal in that they would fertilise and develop into gastrulae, and Lucke concluded from his experiments that the tint given by a pH indicator in a cell was by no means a measure of the pH of its clear protoplasm, but rather of its granules. This must, of course, be admitted ; nevertheless, the average overall pH of a cell is a constant of much interest, and worth investigating.
The method of crushing the cell or cells, and so admitting the indicator to contact with the cell-contents, is an old one, and a great deal of work has been done with it. Its obvious disadvantage is that it does not guard against the effects of cytolysis, but, on the contrary, actually involves them as part of its measurement^. Much work has been done by this type of method on eggs, especially by the Strasburg school. The earliest observation was that of Dernby, who squashed the eggs of Strongylocentrotus lividus in indicator solutions, and obtained a value of />H 6-5 for the interior. He did not, however, follow up this line of work, and the technique was elaborated in much detail by Vies. Vle^' apparatus differed little from what the older microscopists used to call a " compressorium " ; the ^gg of an echinoderm, for instance, was placed in it, surrounded by an indicator solution, the ^gg was then compressed or crushed by turning the screw, until the dye penetrated into the cytoplasm, upon which the pressure was suddenly released, and the outside of the tgg washed with sea water, or colourless solution. Vies found that the eggs of Strongylocentrotus lividus, studied in this way, were yellow to brom thymol blue and to bromcresol purple, but also yellow to methyl red, which showed that the internal pH was between 5 and 6, with an average of about 5-5. No change appeared after fertiHsation, and eggs in segmentation stages gave the same results as the unfertilised ones. Cytolysis, according to Vies, led to a rise in pH, the dye at the edge of the cell becoming purple or blue, instead of yellow, a phenomenon which he attributed to loss of carbon dioxide. At the same time, Reiss published a paper on the internal />H of the nucleus as distinguished from the cytoplasm. Included in it were various determinations by the microcompression method on other eggs, which gave results as follows :
- The same remarks apply to Tchakhotine's attempt to determine intracellular pW by injuring echinoderm eggs in indicator solutions with ultra-violet light.
Species
pH
Investigator
Sea-urchin (Echinus)
4-3
Ashbel
Sea-urchin {Strongylocentrotus lividus)
5-5
Vies
Heart-urchin [Echinocardium cordatum)
5-0
Reiss
Red-currant ascidian {Styelopsis)
4-7
Sea-hare {Aplysia limacina)
4-7
Blood-worm (Arenicola claparedii) ...
6-0
Polychaete worm [Spirographis) ...
6-0
{Nereis)
... 5-8
,, {Sabellaria)
5-0
Gastropod mollusc {Trocho cochlea lineata)
5-2
Clam {Mya)
6-2
Parasitic copepod (Chondracanthus lophii)
... 4-8
Spider-crah {Maia squinada)
... 4-8
For the comparative measures on nucleus and cytoplasm he used the almost transparent eggs of Echinocardium cordatum before they lose their germinative vesicles. He reported that he found the nucleus to take on a deep mauve colour with bromcresol purple, while yet the cytoplasm was distinctly yellow. Bromthymol blue coloured the cytoplasm yellow, the germinative \'esicle yellowish green, and the nucleolus bluish green. The same effects were seen with the unripe eggs oi^ Strongylocentrotus and Sabellaria. Reiss stated that, although the cytoplasm of the egg-cell remained fairly constant with respect to the pa of the external medium, the nucleus was markedly affected by it, and its pH varied in harmony with it. The experiments which supported this view were done on the eggs of Psammechinus milearis and Echinocardium, but they have never been confirmed. Complicated results were also found to follow when anaesthetics were used, but these also have not been repeated, and need not be discussed.
Later, Reiss made a special study of the changes in internal pH taking place in the echinoderm egg during cleavage. He used an apparatus by means of which half the field of the microscope was made to correspond to variable pH indicator tints by means of a series of hollow wedges containing indicator solutions interposed between the microscope and the light source. The accuracy of the method was thus claimed to reach 0-02 pH, but this estimate has seemed much too favourable to other workers on the same subject. With the aid of this apparatus, Reiss found rhythmical variations from pa 5'4 to 5-6 in the cell-interior during the first 250 minutes after fertilisation, i.e. during the first two or three cleavages, and he pointed out that these variations closely agreed with the rhythmical elimination of carbon dioxide found by Vies (see p. 642), but as we have already seen, there is reason for doubting these latter results. Just as Vies' rhythmical results were not confirmed by Gray, so, as will later appear, my wife and I were not able to find evidence in favour of the existence of those described by Reiss.
An alternative colorimetric method to that of microcompression is, of course, that of micro-injection, whether of dissolved or solid indicators. Vies himself, in his first paper on intracellular pH, said, "The microdissection methods of Chambers in vv^hich one injects an indicator directly into the cells, would be evidently the best, but their difficulty hinders their general application". Actually the first intimations of the value of this method were contained in the work of Chambers, who in 1923 injected neutral red solutions into echinoderm eggs, and observed that the tint taken up was always pink, and never more than faintly orange. This direction of investigation was followed up a little later by my wife and myself in a series of papers (Needham & Needham). We injected the appropriate indicators into the eggs of Strongylocentrotus lividus, and found that, although bromthymol blue was always yellow in the cell, bromcresol purple was always purple. This was in contradiction with the results of the Strasburg workers with the microcompression method, as, of course, was our final result of />H 6-5 for the intracellular hydrogen ion concentration. On other eggs treated in the same way we obtained the following values:
Species /)H
Sea-urchin (5<ro«^/ocen<ro<zw ZtwVzw) (unfertilised) ... ... 6-6 «
„ (fertilised) 6-6
Heart-urchin (Echinocardium cordatum) (unfertilised) ... 6-6
Starfish (.4jfen<2j ff/a«a/w) (unfertilised) 6-6
(fertilised) 6-6
,, {Ophiura lacertosa) (unfertilised) ... 6-8
Ascidian {Ascidia jnentula) (unfertilised) ... 6-6
Polychaete worm {Sabellaria alveolata) (unfertilised) . . 6-6
Thus the differences between the kinds of echinoderms were almost imperceptible, and the eggs of a polychaete worm and a tunicate agreed very well with them. All the figures were at least one pYL unit higher than those obtained by the method of microcompression. We attributed this simply to the slighter degree of injury done to the cell by the micro-injection method. At about the same time Schmidttman, using a technique in which she introduced solid particles of indicator into the cells, reported a value of j&H 7-6 to 7-8 for the mammalian egg-cell, taken from ovaries of rats, mice, rabbits, and cats.
This led to an extended controversy, the details of which cannot be given here, but may be found in the original memoirs. Vies and his associates maintained that our readings had been insufficiently corrected, we maintained that tht pH. of the egg-cells studied was in the neighbourhood of 6-5, and that on cytolysis values identical with those obtained by him were found. We regarded cytolysis as an acidproducing process, and considered that his technique was not capable of dem.onstrating the pH of an uncytolysed cell. Thus on injecting bromcresol purple into an unfertilised Strongylocentrotus egg-cell, for instance, one sees what we called a "purple puff" which lasts for some seconds, then giving way to a greenish tint, which soon turns yellow. But on microcompression of such an egg, according to Vies, the first colour seen in the cell is yellow or yellowish green, so that, in our opinion. Vies was never observing uncytolysed eggs at all.
In support of our view that the intracellular reaction is near the neutral point, and that cytolysis liberates acid substances which interfered with Vies' method, there are many observations. Since, as we have seen, Warburg has shown that the lactic-acid-producing mechanism is not peculiar to muscle, but exists in all tissues, the probability of lactic acid being produced in cytolysing eggs is very considerable. Again, any vacuoles would naturally be expected to burst when the cell is squashed, and, since Greenwood showed as long ago as 1894 that the food-vacuoles of protozoa are distinctly acid, there is obvious danger from that source. Recently Rowland has found a pYi of 4-3 in the digestive vacuoles of Actinosphaerium. But, further, the researches of Parat and his school have shown that the so-called Golgi apparatus is in all probability a series of intraprotoplasmic spaces, a vacuome, which contains an acid liquid, and is filled up by the metallic reagents usually employed to demonstrate it. For Triton marmoratus egg-cells Parat gives a />H of 7-2-7'3 for the protoplasm, but of G-S-G-g for the vacuome: for Ascidia mentula egg-cells 6-8 and 5-0 respectively (vital staining). (Only four complete studies of the behaviour of the Golgi apparatus during embryonic development exist: Gatenby and Hirschler on Limnaea stagnalis, Nihoul on the rabbit, Parat on the nudibranch molluscs Aplysia and Polycera.) Finally, Drzwina & Bohn have described a phenomenon which may be called "infectious cytolysis". If a number of small animals, such as planaria, or of eggs, are placed in a small space, it is found that the cytolysis of a few sets all the rest cytolysing, and these workers found that a culture of Convoluta by its cytolysis lowered the pB. of its culture medium from 7-0 to 4-4 in two minutes. Similar observations were made by Rebello and by Drastich. In our own experiments we observed that when amoebae cytolysed, their internal />H went down from 7-6 to about 5-0, but when the marine egg-cells cytolysed their pH descended further and more rapidly from 6-6 to about 4-5. This has since been confirmed by Reznikov & Pollack. On these grounds we concluded that our more alkaline figures were more accurate than Vies' acid ones. We laid some emphasis on the fact that we observed no change in the intracellular /?H on fertilisation, and that, although we micro-injected blastomeres up to the morula stage, we never found any perceptible variation during the cleavage period. The intercellular fluid in the i6-cell stage and the liquid filling the blastocoele cavity we found to be at least as alkaline as pYi 7-3. This was almost exactly the same figure as one previously obtained for the blastocoele of Pomatoceros by Horstadius.
Certain other points also emerged from our work on these marine invertebrate eggs. If the egg-cells of Asterias were fertilised with concentrated sperm suspensions, and afterwards kept in conditions of bad oxygenation, a high percentage of abnormal forms made their appearance, protruding bulbs of protoplasm, dumb-bell shapes, abnormal divisions, etc. But in all these cases the intracellular j^H was normal, and remained so until cytolysis set in, when it was, of course, lowered to/?H 4-5, or below. Again, in injections of the 2-cell stage in Strongylocentrotus, one blastomere would be quite coloured with the dye used, and would even cytolyse before its fellow showed any abnormality. There was no connection between them. Then we found that in acid sea water of p¥L 6-o the eggs would remain for at least 2 hours without showing any change in internal />H, maintaining independence thus against a change of 2-4 pH units in their environment.
Subsequent micro-injection work has uniformly confirmed our findings as regards intracellular pH. Rapkine & Wurmser injected dissolved indicators into the nucleus and the cytoplasm separately of the egg-cells of Strongylocentrotus lividus and Asterias rubens, and could not distinguish any difference between the pH. Both nucleus and cytoplasm were in the close proximity of pH 7-0. Chambers & Pollack, however, did obtain a certain difference between the nucleus and cytoplasm in the case of Arbacia eggs, getting values of 7-6-7-8 for the nucleus, 6'6-6-8 for the cytoplasm, and 5-4-5'6 for the pH of cytolysis. They found, just as we had, that cytolysed material in time assumes the /?H of the surrounding sea water; an observation which probably explains the tendency towards alkalinity which Vies had associated with cytolysis. Injury to the nucleus did not affect its j&H, but the spherical nuclear remnant persisting after injury gradually assumed the />H of the environment. Curiously, an indicator for which the egg was normally impermeable could penetrate into it through a tear in the surface if the environment was more acid than normal. Perhaps the plasmalemma coat of the protoplasm does not form so completely in an acid medium.
Our observations on the j&H of the blastocoele cavity were greatly extended by Rapkine & Prenant, who in 1925 followed the course of events in detail. To begin with, in the blastula of Strongylocentrotus lividus, thepH. (ascertained by micro-injection of dissolved indicators) was between 7-0 and 7-3, but a little after gastrulation, as soon as the primitive mesenchyme cells appeared, it rose through 8-o, at which point the spicules were first formed, to 8-5, after which it gradually descended again to its original value of rather less than the surrounding sea water. It was evident that the rise and fall of the curve (shown in Fig. 208) was associated with the process of deposition of calcium for the spicules. pH 8 had already been noted by Prenant to be the most favourable hydrogen ion concentration in vitro for the deposition of calcite, and it is known that the spicules of echinoderms are of this mineralogical form. This work was repeated on the eggs of Echinocardium cordatum, with the result that a precisely similar curve was found. Rapkine & Prenant pictured a selective absorption of the carbon dioxide produced by the organism as a whole by the mesenchyme cells for the manufacture of calcium carbonate, a process which might well be expected to make the liquid of the blastocoele cavity more alkahne than usual. Next Bouxin found that a fall of pH irrespective of the acid employed would produce in Strongylocentrotus lividus larvae a retardation of skeleton formation or a complete stoppage, or even if the p¥L was lowered below 6-4 a regression of spicules already formed. Rapkine & Bouxin accordingly micro-injected indicators into the blastocoele cavity at different external hydrogen ion concentrations, and in fact found that when the exterior /?H went down to 6 that of the blastocoele cavity also went down, and almost as far, keeping above it, however, to the extent of two or three tenths of ^^h a />H unit. In the extreme cases there seemed, then, to be an actual solution of the spicule. Rapkine & Bouxin found that during this process the j&H of the liquid in the blastocoele cavity was maintained rather stable at ^^' ^° "
/>H 6*25, and they suggested that the regression of the skeleton might to a certain extent be considered as a defence mechanism in view of the fact that death invariably occurred when the blastocoele pYi had reached 6-o. In the case of younger embryos, where the mesenchyme cells had just appeared, a small fall in external />H causes a retardation or an inhibition of skeleton formation, while the j&H remains at about 7-4; thus the mesenchyme cells are removing carbon dioxide, but the external acidity prevents the j&H rising.
Contributory evidence showing the retention of carbon dioxide during spicule formation was provided by Vies & Gex, who placed developing echinoderm eggs in a small chamber surrounded by a dilute indicator, and then examined the system from time to time spectrophotometrically. They obtained the figure shown in Fig. 209, from which it can be seen that during the first 4 hours the />H fell steadily (N.B. no sign of rhythmic change), but that between the 4th and the 6th hour there was a kink in the curve, indicating a retention of carbon dioxide, and even a slight absorption of it. This corresponded exactly with the time of skeleton formation, as the other curve on the graph shows, relating as it does number of embryos with spicules to time. More recently, however, the micro-injection work of Rapkine & Prenant has been repeated by Chambers &
Fig. 209. Experimental values • — Corrected values
Pollack, who have not been able to find similar results. Using the blastulae of Asterias forbesii, Echinarachnius parma, and Arbacia punctulata, they micro-injected dissolved indicators into the blastocoele cavity, and in all cases found its pH to be the same as that of the external sea water. If the latter was brought down to pYi 5 or 6, the liquid in the blastocoele cavity also gave a result of j&H 5 or 6. Chambers & Pollack claimed that, by using very slender pipettes and inserting them between rather than through the cells of the wall of the blastula, the pH of 8-4 was found for the interior fluid, and that injury to the cells surrounding it accounted for the lower hydrogen ion concentrations found by Rapkine & Prenant^. Chambers & Pollack concluded, therefore, that the pH of the liquid filling the cavity is until metamorphosis exactly the same as that of the sea water in which the embryos are placed. The regularity of the curve obtained by Rapkine & Prenant is, of course, an argument against these criticisms, but, in view of the difference of opinion, similar experiments should certainly be undertaken again.
Electrometric measurements of j&H were begun by Vies, Reiss & Vellinger in 1924, who made a thick suspension of eggs, freezing them solid very rapidly, pounding up the hard mass, and then placing the electrodes in contact with it as it melted. Unfortunately this procedure did not give one definite potentiometer reading, but a whole ascending curve, so that it was necessary to choose a time for taking the reading. Vies, Reiss & Vellinger chose the moment when the thermometer indicated 0°, and calculating from that they obtained for normal Strongplocentrotus eggs an average value of j&H 5-3, or for 18° 5-1. For eggs from which the jellies had been first of all removed by potassium cyanide the value was 6-3 for 18°. The three investigators explained this more alkaline figure as being due to the easier escape of carbon dioxide in the case of the dejellified eggs, though, as the whole system was one more or less homogeneous paste, it is difficult to understand this argument. In general, they concluded that the electrometric method gave results which fully confirmed their work with the microcompression colorimetric method. This was doubtless true, but it seemed to other workers that the reason was mainly because the principal source of error, i.e. cytolysis and acid production, was the same. Injury is almost certainly done to cells on freezing by the formation of ice crystals, and even if the acid production of cytolysis is in abeyance at the low temperature, it will come into play as the temperature rises, even in all probability before 0° is reached.
Vellinger, however, has continued researches in this direction, and the method has been used by the Strasburg school in the case of many types of cells other than eggs and embryos. Vellinger used a temperature of — 60° at which to crush the eggs, and got the same results as before, but this improvement does not make the "puree" method inherently more satisfactory. He calculated the intracellular />H of Strongylocentrotus lividus eggs to be 5*8-5-9, and of Arbacia equituberculata 5-0-5-2. But as Chambers & Pollack afterwards pointed out, the worst feature of this method is that the potentiometer gives a curve as the "puree" melts, and the exact point which is chosen for the reading rests on arguments no better than could be adduced for taking it at another point.
- 1 But would the cytolysis of two or three cells suffice to acidify the whole blastocoele cavity?
The other type of electrometric method, where electrodes are actually inserted into the tgg, has not been so much used. Bodine, using a micro-electrode of his own design, requiring o-oi c.c. of material, measured the/>H of the egg-contents of Fundulus heteroclitus. The resulting mean average pH was 6-39, and the limits were 6-i and 6-8. No change was to be found at fertilisation or afterwards up to 17 days' development, except that the results after fertilisation seemed to come more constant than those before. Death brought about a very acid reaction, which lowered the j&H as far as 4-4. Placed in hydrochloric acid solutions of pH 4-3, the egg-contents remained quite unaffected for at least 100 minutes, but eventually changes took place inside the egg. As far as this went, the unfertilised eggs were less resistant than the fertilised ones, but there was no change in the relative resistance during subsequent development. Work on Fundulus was continued by Armstrong (but by microinjection of indicators). The subchorionic space was />H 8-4 ( = sea water) and if the eggs were put into distilled water, descended to 5-6 in 18 hours. The pericardial cavity was still 8-4 after that time, however, as were the brain vesicles. The pH. of the yolk was always close to 6-0.
Bodine's work was hardly a measurement of protoplasmic />H, in view of the highly lecithic nature of the egg of the minnow, but a number of interesting results on frog eggs were supplied by the work of Buytendijk & Woerdemann. They found that the hydrogen electrode and the quinhydrone electrode were for various reasons inapplicable to the determination of the intracellular j&H, so they made use of an antimony electrode designed specially for the purpose. The micro-electrodes of Ettisch & Peterfi and of Taylor & Gelfan had not been suitable for /)H determinations, but they were modified to carry Buytendijk's antimony electrode. This metal, enclosed in a glass micropipette, proved very useful, for, as has long been known, it gives a potential difference depending regularly on the hydrogen ion concentration of the liquid with which it is in contact. Placed in one blastomere of an amphibian egg, the electrode was made to register graphically changes in pH. The eggs used were those of Amblystoma, Triton taeniatus and Rana temporaria. The main results were as follows:
Ovarial eggs ... ... 7'2
Fertilised eggs ... ... 8-5
2 -cell stage 8-5
4-cell stage ... ... ... 8-05
32-cell stage ... ... 7-9
64-cell stage ... ... 7*9
As regards the intracellular pYi, the value of 7-2 for the ovarial eggs was almost exactly the same as that of the adult blood, i.e. 7-35, a fact which recalls the osmotic pressure measurements of Backmann and his collaborators. It was not in agreement, however, with Reiss' value of 6-o for the unfertilised frog's tgg, by the microcompression method. Newly laid eggs surrounded by solutions of />H 5-9 or 7-7 showed no change at all in intracellular pH, at any rate over a comparatively short period. Buytendijk & Woerdemann inserted their electrode into one blastomere after another in the same embryo without ever finding more than minute differences in p\i. Cytolysis, they found, led invariably to a decrease in p¥L, obviously due to acid production, thus agreeing with our results and those of Chambers & Pollack on echinoderm eggs. The fact that the eggs which had been pierced many times with the antimony electrode still continued to divide normally indicated, they felt, that it was a very harmless instrument.
They emphasised the fact that the amphibian eggs registered a very marked change in/?H on fertilisation, contrary to what had been found for echinoderm and teleost eggs. Their rise of 1-25 j&H units then was in agreement with Reiss' rise of o-4/>H units. But what was most noticeable about their values was that they were all distinctly higher than those of any other investigators, and while this may be due to the special material they employed, it is also very likely that of all the methods which ha\e been used theirs does least injury to the cell, and so causes least production of acid. It would be extremely interesting to use the antimony micro-electrode on echinoderm eggs. For later stages the following interesting values were obtained:
Table 98. Triton taeniatus.
pa
Blastula External cell layer y-G-y-S
Blastocoele liquid 8-4-8-6
Gastrula (beginning) ... Ectoderm y-G-y-Q
Gastrula (late) ... ... Ectoderm 7-6
Endoderm ("Urdarm") 8-i
Neurula ... ... ... Ectoderm cells 6-9-7-0
Cells of neural tube 6-8
Endoderm ("Urdarm") 8-i
Endoderm (yolk-mass) 6-g-7-o
An interesting research on the eggs of Arbacia was that of Vies & Vellinger, who made spectrophotometric observations on the pigment normally contained in these cells. This involved no interference with the material at all, and only required a preliminary isolation of the pigment and a study of its properties in vitro. It must be remembered that the behaviour of the pigment in the cell as a pH. indicator may only show the hydrogen ion concentration of a very limited phase, in which the indicator happens to exist. Vies & Vellinger attempted to overcome some of these difficulties by comparing the colour change of the indicator (their "Arbacine" was probably identical with McClendon's "Echinochrome") [a] in alcoholic solution and {b) unremoved from a thawing "puree" of eggs. They showed that the colour change from orange through violet to yellow took place according to j&H in much the same way in the two cases. By a comparison of the spectrum of the pigment when in the intact cell with the various spectra of the pigment under known conditions of />H, they ascertained that the former corresponded to a />H of about 5-5, from which they concluded that this represented the pYi of the cytoplasmic elements or phases where the pigment was present. Spectrophotometric curves confirmed the results obtained by simply looking at the spectra, save that there was evidence of two pigments, one with two maxima and one with one. The pigment of the Arbacia egg is known, according to the work of Heilbrunn and many others, to be localised in certain definite elements. This means that what is probably the best possible method of studying the intracellular hydrogen ion concentration has not so far been able to give a value for the inside of a cell as a whole, or for what we roughly call protoplasm. It is to be feared that it will always be of less general use than the micro-electrodes, because so many egg-cells are loaded with yolk and opaque substances, and so few possess a pigment which is a natural indicator,
A certain number of experiments have been made in which the effect on development of changing the pH of the external medium has been studied. Vies, Dragoiu & Rose, following again their conception of "travail d'arret", determined the j^H necessary for complete cessation of cleavage in echinoderm eggs. In the case of Strongplocentrotus lividus the percentage of eggs having accomplished the cleavage in question remained high and constant until />H 5-2 was reached, but between 5-2 and 4-9 it decreased by almost 100 per cent. A second paper by Dragoiu, Vies & Rose studied the incidence of cytological abnormalities in the eggs submitted to abnormal hydrogen ion concentrations, establishing the expected result that the more abnormal the pH the more rapidly the abnormahties occurred. Here again, complete curves on a graph were plotted from only two points in each case. Now, although no effect on the number of cleavages in a given time had been observed between the normal /?H (8-4) and 5-2, it was possible that a small effect had been produced. Labbe argued that this would be better shown over a longer period, so he determined the time taken to reach a definite stage in some polychaete worm embryos, obtaining figures as follows : ^. , - • .
^ ^ Time taken to reach a given stage
in hours from fertiHsation at
pH 8-4 (normal) pH 8-1
Sabellaria alveolata ... ... ... 27 45
Halosydna gelatinosa ... ... 18 28
S . alveolata y. H . gelatinosa ... ... 19 3°
Evidently a comparatively small change in /?H shows a marked effect on developmental time over a long period.
Clowes & Smith and Smith & Clowes, in a series of interesting papers, studied the effect of j&H on various factors in development, such as the ageing of unfertilised Arbacia, Asterias and Chaetopterus eggs, the artificial activation of Chaetopterus eggs, and the development of normally fertiHsed Arbacia and Asterias eggs. Loeb's early work with Arbacia eggs had shown that addition of acid to sea water was always retarding in its action, but that small amounts of alkaH exerted an accelerating action. This accelerating action was not operative before the formation of the blastula, but only between that stage and the stage of the pluteus. Excessive amounts of alkali had, of course, an injurious effect, and the maximum was obtained when 1-75 CO. of JV/io NaOH were added to 100 c.c. of sea water. He attempted to raise the eggs of Strongylocentrotus from fertiHsation in neutral Ringer's solution without success, but found that with the addition of a little potassium hydroxide or sodium bicarbonate it was possible to do so. He concluded that a neutral or faintly alkaUne solution was necessary for normal development, and subsequent experiments by Herbst and by Peter only confirmed this view. Moore,
Roaf & Whitley, investigating Echinus eggs, found exactly the same relationships — the smallest amount of acid inhibited growth and cleavage, but alkali had first a stimulatory and then an inhibitory effect. In a later paper, Whitley could not find any evidence of the favourable effect of mildly alkaHne hydrogen ion concentrations in the case of the eggs of the plaice, Pleuronectes platessa, but reported that a change of />H towards the acid side was much more fatal than one towards the alkaline side. Loeb's work with Arbacia eggs was repeated by Glaser, who also observed the stimulatory effect of small amounts of alkah, but emphasised that this is hmited to the postblastula stages, for the cleavage-rate at the beginning may even be a little retarded. Medes studied the morphology of the plutei raised from solutions of varying acidity. Finally Richards observed acceleration of the early cleavage stages of the opisthobranch Haminea virescens in sea water to which sodium hydroxide or potassium hydroxide had been added. None of these workers had controlled the/>H, so Smith & Clowes undertook to do so, raising these numerous isolated fragments on to a quantitative basis. Their results are shown in Fig. 210, where the percentage development (number of cleavages per tgg) is shown plotted against the pH. of the sea water, normal development atj&H 8-15 being taken as 100. The sharp drop between />H 4-8 and 5-4 equates well with the results of Vies, Dragoiu & Rose. The slight effect of hydrogen ion concentrations above normal in accelerating division is well shown on the curves ; as the /?H is raised it soon gives place to the retarding effect which brings the percentage development down to zero by the time />H 10 is reached. The striking thing is that the limiting hydrogen ion concentrations are characterised not by a gradual but by an abrupt inhibition of development, while between them it is essentially unimpaired. These results were afterwards repeated and confirmed by Gellhorn.
According to Smeleva and McCoy, nematode eggs are independent of external pH over a very wide range ; an interesting fact in view of their remarkably impermeable membranes (see p. 327).
6-2. The pH of Terrestrial Eggs
As regards terrestrial eggs, those of birds have been most investigated, but we possess some figures due to Fink for the />H of the eggcontents of some insects, colorimetrically estimated, as follows :
Colorado potato-beetle {Leptimtarsa decemlineata)
,, peach-beetle {Cotinis nitida) ... Japanese beetle {Popillia japonica) Squash ladybird {Epilachna borealis) Seedcorn maggot {Hylemyia cilicrura) ... Squash bug {Anasa tristis)
Thus in some cases there was no change, and in others there was a certain rise towards the alkaline side as development proceeded.
We may now take up the discussion of the total acidity and the pYi of the various parts of the bird's ^gg, and the changes which it undergoes during the course of development. The classical paper on
N E II 55
Soon after
Just before
laying
hatching
6-8
6-8
6-2
7-1
7-1
7-1
5-9
U
5-9
6-2
6-4
this subject is that of Aggazzotti, but earlier workers made some observations of the kind. Thus in 1863 Davy reported that he had found in many kinds of birds' eggs that the albumen was always alkaline and the yolk acid. In 1884 Tarchanov, in the course of his work on " Tataeiweiss " already referred to (p. 272), measured the titratable acidity of the egg-whites of various eggs, obtaining the following results : .„ ,. .
Alkalinity expressed as grams potassium hydroxide in per cent, of dry weight Nidicolous Raven (fresh) 4-9
„ (dev. 2 days) 1-4
,, (still more) o-8
Pigeon (fresh) 4-7
,, (i week) 2-8
Nidifugous Hen (fresh) 7-1
,, (i week) 4-7
,, (1^ weeks) 2-7
,, (2 weeks) 2-3
Thus the titratable acidity was greater in the case of the whites of nidicolous birds than in those of nidifugous ones, and in all cases it increased as development proceeded. This has often since been confirmed. A solitary figure of Reiss' is available for the yolk and the white of an elasmobranch egg, pH 5-6-6'0 in the case o^ Scyllium canicula}.
Aggazzotti's careful work on the hen's tgg, published in 191 3, involved many measurements, both of pH. and titratable acidity, which are incorporated in Figs. 211 and 212. The measurements of />H were all made electrometrically. Taking first the graph which shows the />H, it can at once be seen that Davy had been quite right. The yolks of the eggs investigated by Aggazzotti had an average pYl before incubation of 4-5, and the egg-whites one of 8-3. If the eggs were not incubated, this hydrogen ion concentration remained unaltered, as is shown on the graph by the dotted lines, and no change took place over an even longer period. If incubation occurred, however, there were marked changes in the fertile egg. The j&H of the yolk rises steadily, attaining /?H 6 about the loth day of development and neutrality by about the i6th, while that of the white equally regularly falls, reaching neutrality on the loth and pYi 6 on the 15th day of incubation. There is thus a cross-over point when 50 per cent, of development has been completed, and after that the white is more acid than the yolk, though neither is as far from the neutral point as at the beginning. As for the allantoic liquid, Aggazzotti did not collect many figures for it, but its pH seemed to follow a curve convex to the abscissa, but always near neutrality, while that of the amniotic fluid wavered around neutrality until the nth day, after
- Nothing is known about the pH of reptile eggs but their egg-white is non-coagulable, like that of nidicolous birds (Deraniyagala) .
o White, Aggazzobti
a White, Vladimirov
A White (fchick),Baytendijk
V White (thm), „
O White,Healygj Peter
White,Gue_ylard^Portier
O All., .= " »
• Yolk, Aggazzotti ■ Yolk,Buytendijk
♦ Yolk,Healy8j Peter
YoikjGuej/lard^Portier ® Amniotic f laidjAggeizzotti B »> 5> ,Bayfcend(jk o Al^Aggazzotti <^>
O
Days
12 3 4
J 1 L
5 6
L
I 72 I 120 I 168 I
9 10 11 12 1314 15 16 17 1819 '20 21 J \ 1 \ i 1 \ 1 1 1 1 \ L
I 312 I 360 1 408 I 456 I
24 I 72 I 120 I 168 I 216 | 264 I 312 I 360 I 408 I 456 I 504 48 96 144 192 240 288 336 384 432 480 Hoars Fig. 211.
which it plunged rather suddenly into the acid region. Fig. 2 1 2, which shows the total acidity expressed in c.c. of JV/ioo H2SO4 or NaOH required to neutralise to a-naphtholphthalein i c.c. of yolk or white diluted to 3 c.c. with i per cent, sodium chloride solution, gives a very similar picture. Just as the pH of the yolk rises during incubation, so there appears to be less free acid there, and just as the p¥L of the white falls, so there is more free acid
present in it. The amniotic and allantoic figures present analogous curves. Aggazzotti found that if infertile eggs were incubated there was no change at all in the pH of their white, but that the yolk pB. rose just as in the fertile egg, but not so far, i.e. up to pH 6-2 or thereabouts, and never further. This presents an analogy with BelHni's findings on the viscosity of incubated infertile and fertile eggs, where the same change seemed to go on in the yolk of the infertile egg, but to a less extent, while the white was far less affected. It is probable that these phenomena are due to the presence of enzymes in the yolk and not in the white, a statement generally true, as the Section devoted to enzymes will show. These enzymes will naturally begin to work at the beginning of incubation irrespective of whether the egg is fertile, and will produce some of the changes associated with development, but it is reasonable to suppose that the embryo exerts a furthering influence upon their activity. This was indeed the explanation adopted by Aggazzotti.
The significance of all these changes is not easy to understand. It must, for instance, be important that at the end of development the embryo is surrounded on all sides by liquids of acid reaction. The fact that the slope of the two curves (the descending one of the white and the ascending one of the yolk) is very much alike, led Aggazzotti to suggest that there was some simple relation between them, such as a transfer of hydrogen ions from the yolk to the white. But it is certain that events are more complicated than that. One very interesting fact appears, if the behaviour of the amniotic fluid in Fig. 211 is compared with that in Fig. 212, for it is then seen that, though the final pH of the amniotic fluid is much the same as that of the yolk at the beginning of development, their total acidities are by no means the same, the former being only half the latter. It is obvious, therefore, that the dissociation constants of the acids responsible are very different, being much greater in the case of the final amnios pH than in that of the initial yolk.
Fig. 21Q.
Since the time of Aggazzotti several workers have occupied themselves with the same problems. Healy & Peter examined the yolk and white during the early part of development with the special intention of finding what buffers were there. They obtained figures of j&H 6-2 to 6-6 for the yolk and 8-2 to 8-4 for the white, and they confirmed the observation of Aggazzotti that no change took place over a long period if the eggs were not incubated. They estimated the alkali reserve of the white by titrating to different end points with jV/io HCl. Thus the following figures,
White
Yolk
Phenolphthalein Methyl orange Phenolphthalein c.c. jV/ioHCl c.c. jV/ioHCl c.c. ^'/lo NaOH
Incubation of 3 days 1-4 15-3 5-2
,, 6 days 1-7 14-8 12-9
indicated that the main part of the alkali reserve was in the form of sodium and potassium carbonate. Parlov confirmed Aggazzotti's results on the egg white, using the ingenious method of boring a hole in the shell and withdrawing samples each day of incubation. The/>H fell from g-o to 7-0. A good deal of evidence exists that the high alkalinity of the egg-white of the new-laid egg is due to the escape of COgfrom it (see Fig. 152)^. Thus Buckner & Martin found oviducal egg-white to be at p¥L 6-7 though after laying the usual figure of 9-0 was obtainable, and similar results were reported by Romanov & Romanov in a complete research which gave curves resembling closely those in Fig. 211. The alkalinity of the new-laid egg-white is connected with its bactericidal properties for a discussion of which see Section 19-3.
Vladimirov also measured electrically the pH of the white of the hen's egg during its development. His figures are shown in Fig, 211 plotted on the same graph as those of Aggazzotti. It is interesting that the fall in pH takes place at exactly the same rate as found by Aggazzotti, but always about one pH unit higher than those of the Italian worker. The still more recent figures of Buytendijk & Woerdemann agree with those of Vladimirov rather than with those of Aggazzotti, a fact which is all the more
1 In an atmosphere containing 1 2 % COj the pH of the egg-white remains constant at 7-8 for the first six days after laying, showing neither the usual rapid rise nor the usual slow fall (Fig. 211). And the pH may be varied at will by adjusting the external carbon dioxide concentration (Romanov & Romanov).
Fig. 213.
Striking because their yolk figures also rise parallel with the earlier ones, only again one pH unit higher. All these relations are shown in Fig. 211. The cross-over between yolk and white occurs at almost the same place in all cases.
Buytendijk & Woerdemann's figures, given in Fig. 211, show a good agreement with the results of the previous observers, which is rather gratifying in view of the good technique used by them. A point of much interest is that Buytendijk & Woerdemann measured the pH of the less viscous and the more viscous parts of the white separately, and found that, while the pB. of the white as a whole was falling, that of the former part of it fell more rapidly than that of the latter part. Doubtless this is due to the faster diffusion of the responsible acids into the more Hquid portions. The electrometric work of Gueylard & Portier has also supplied a few figures for yolk, white and amniotic fluid, which have been incorporated in Fig. 211. Perhaps they indicate a late acidification of the allantoic as well as the amniotic fluid.
Pigorini's figures for total acidity of the silkworm egg may be mentioned here (see Fig. 213).
We may now pass to the measurement of the pH of the embryonic cells. For a time it was thought that results of value could be obtained by crushing the tissues in some convenient apparatus, and then estimating the pH of the "Pressaft" colorimetrically or electro
Fig. 214.
metrically. Thus Gueylard & Portier employed this method, obtaining the curve shown in Fig. 214, but not putting forward any explanation for the sharp trough passed through about the 15th day. A much more complete piece of work, which involved the crushing of the cells before they were brought into contact with the indicator, was that of Murray. His figures are given beside those of Gueylard & Portier in Fig. 214, and it is unfortunate to note that, although his period was better investigated than theirs, the two do not overlap completely, so that we cannot tell whether Murray would have got the low values about the 15th day if he had gone on. He found that a regular S-shaped curve (dotted in Fig. 2 1 4) would fit them, but it is unUkely that this was more than mere coincidence. As for the points of Friedheim and of Yaoi, they disagree with both the other sets.
Fig. 215.
Murray, however, estimated some other entities as well as the pH by crushing the cells, and it will be best to describe his results here. Fig. 2 1 5 shows the molar concentration of chlorides (determined by the Van Slyke method) in the embryonic tissues related to age, and Fig. 216 the molar concentration of total carbonic acid similarly plotted. Murray was inclined to correlate the increasing acidity of the tissues with the accumulation of the carbon dioxide of catabolism, but this, though a sufficient explanation for his own few figures, does not cover, as it stands, those of Gueylard & Portier, still less those of Friedheim and of Yaoi. Murray called attention, however, to other factors which will obviously have to be considered with relation to the pH. of the embryonic cells, i.e. the functional efficiency of the systems of the organism, such as the circulation, whose function it is to remove the carbon dioxide produced in metabolism, and secondly the process of ossification, which may affect the concentration of carbonates in the tissues. Since the metabohc rate decreases with age, it alone can hardly be held responsible for the increasing acidity. Cohn & Wile showed that during the early part of development there is a marked increase in the rate and regularity of the cardiac contraction, which might explain the apparent constancy of the tissue />H before the loth day. Murray related the S-shaped curve of his j&H data to the gradient existing between the carbonates in the shell and in the bones, and he supposed that the entry of calcium carbonate from the shell would lead to the increasing concentration of carbonate in the tissues which he found experimentally. He suggested that it might be possible to find by calculation that amount of carbonate in the functioning tissues. It was known that the concentration of protein in the embryonic body rises during incubation, so Murray suggested that, as proteins would act as anions at the pH found, they would replace the diminishing chloride. Unfortunately, we may be quite sure that the pH. as found by the crushing method employed by Murray is not that of the uninjured cells, so that this calculation loses most of its force. Murray's observation, however, that the point of greatest increment on the rising protein curve came some 4 or 5 days after the point of greatest decrement on the falling chloride curve, remains quite true. "The results of our observations", said Murray, "which show that the electrolytes probably change several days before the protein, and the latter several days before the fat, lead to the conclusion that the processes of chemical differentiation are not to be described by a concept of dynamic equilibrium but rather by a notion of 'follow my leader'. The leader in this case is presumably the most rapidly permeating, reactive, and mobile molecule and tentatively we ascribe this role to the CO2 of metabolism." What does this mean?
Fig. 216.
Parallel experiments to these were made by Woglom on the embryo of the rat, using a technique which included the fine mincing of the tissue, and the electrometric determination of the pH on the resulting cell-emulsion. He got values of /?H 7-04 to 7-36, with an average at pH 7-14. Then Ruzicka, who did not give any details of the method he employed, but presumably worked with minced or crushed cells, obtained the following series :
Table 99.
Frog (Ranafusca) pH
Unfertilised eggs ... ... ... ... 6-6
Morulae ... ... ... ... ... 6-i-6*2
Gastrulae ... ... ... ... ... 6-4
First appearance of medullary plate... ... 6'2
First appearance of tail bud ... ... ... 6*o— 6'2
Larva 6 mm. long ... ... ... ... 6-4
Appearance of external gills ... ... ... 6-7
Disappearance of external gills ... ... y-o-y-l
Larva 15 mm. long ... ... ... ... 6-8
Larva 22-7 mm. long ... ... ... ... 6-9
First appearance of hind legs ... ... 7-8
Completely formed hind legs... ... ... 7-0
Immediately after metamorphosis ... ... 7-2
Sexually mature frogs ... ... ... 7'5-7'9
He pointed out that a continual rise took place, a change from the acid side to the alkaUne side, and suggested that it was an approach towards the isoelectric point of the tissue proteins.
Much more satisfactory was the work of Buytendijk & Woerdemann with their antimony micro-electrodes. Using them in the way described above, they obtained the following results :
Table lOO.
Day of development of the chick emb
4
7 13
Optic vesicle
6-75
7-3-7-4 7-7
Brain rudiment
6-7
7-0-7-4 7-0
Myotomes
7-0
7-5-7-6 7-0
Liver
- 7-2
Heart
—
- 6-8
Stomach contents
—
- 5-8
Duodenum contents ...
—
Ileum contents
—
— 6-8
Leg muscle
—
6-9
Some difference was made according to how long the estimations were done after the cessation of the circulation. On the whole, the values for the 13th day were lower than those for the 7th day, but higher than the figure ob
Cohn.Mirsky ^ Porosovski
(Glass electrode)
) redoced » oxygenated
t
tained by Murray and Gueylard & Portier.
Another aspect of this work is the />H of embryonic blood. Murray made no estimations of this, but Cohn & Mirsky and Cohn, Mirsky & Porosovski afterwards made complete measurements for the chick embryo at all stages by ' pjg 217.
means of the glass electrode.
Between the 6th and the 8th days the blood was relatively acid, and from the 9th to the 15th days there was a plateau just on the alkaline side of neutrality, after which the rise towards the alkaHne side was continued, to reach the adult level at the time of hatching. Experiments on the blood of the developing cat embryo gave very similar results. It is obvious that the course taken by the blood />H in the chick agrees closely with that of the /'H of the yolk. There is also agreement here with the fragmentary resuhs of Gueylard & Portier, who found blood hydrogen ion concentrations ranging from 8-4 to 8-1 on the last 2 or 3 days of the chick's development. The few figures collected by Hajek would seem to indicate that the rise to the adult pH level in human blood from the acid side has attained completeness at birth.
Millet has worked on the blood and tissues of the embryonic rabbit, using the glass electrode, and Mendeleef has done similar experiments on the blood of the guinea-pig, using colorimetric
Fig. 21
Fig. 219.
methods. From Figs. 218 and 219 it can be seen that in each case there is a passage from the acid side to slightly above neutrality, and this agrees with the work of Ruzicka; Buytendijk & Woerdemann; and Cohn & Mirsky. But there is Httle to show that these curves may not be due to a steady increase in the amount of blood taken for sample, rather than to a real increase in pH, and as yet too much emphasis must not be laid upon any of these conclusions.
6-3. pH in Embryonic Life
Closely related to the />H is another factor, less generally used by biologists, the rH. Oxidation-reduction processes, like acid-base equilibria, have an intensity as well as a capacity factor. Just as we may speak of the hydrogen ion concentration, distinguishing it from the total amount of acid or alkali present, ascertainable by a titration which mobilises the factor titrated as fast as it is used up until no more is left — so we may speak of the oxidation-reduction potential, meaning the intensity of electron transfer in the system, as opposed to the total quantity of oxidants or reductants present. These conceptions apply, strictly speaking, only to systems which are reversible, while the living cell is a complex association of oxidationreduction systems, some of which are probably reversible, and some of which are not. Nevertheless, the application of the conception of oxidation-reduction potential to the processes occurring in the living cell has justified itself by its results. The living cell cannot be thought of as a simple reversible system, and true equilibrium must be sharply distinguished from a steady balanced state maintained at its characteristic level by the velocities of a chain of reactions. The latter condition is what is found in the living cell, which is balanced very steadily at its characteristic level of oxidationreduction intensity. Between the entering hydrogen acceptor, oxygen, of high rH, and the reducing systems such as glutathione and xanthine oxidase, of low rH, the cell maintains its overall rH closely around oxidation-reduction neutrality in ordinary aerobic conditions. These interesting problems cannot be treated here in detail, but a full account of them will be found in the series of experimental papers due to Mansfield Clark and his collaborators. The reviews of Clark; Dixon; Conant; Michaelis and Wurmser deal with the theoretical aspects of the application of rH to biological problems, and the review of Needham & Needham should be consulted for an account of what has actually been done in this direction. It must suffice to say here that, just as ihepH is the negative logarithm of the hydrogen ion concentration, so the rH is the negative logarithm of the pressure of hydrogen gas in a platinum electrode in equilibrium with the given system, and to note that the behaviour of strong and weak acids and bases, buffers, indicators, etc., all find a counterpart in oxidation-reduction equilibria.
The earliest determinations of the rH of biological systems by means of rH indicators, i.e. dyes whose oxidation-reduction potentials at all stages of decolorisation to the leuco-bases had been accurately ascertained in vitro, were made by Clark. In 1 925 my wife and I began a series of experiments in which these indicators (both completely oxidised and completely reduced) were micro-injected into single cells.
As regards the rH of the egg-cell, we reported that, in Strongylocentrotus lividus, Asterias glacialis, Ophiura lacertosa, Echinocardium cordatum, Sabellaria alveolata, and Ascidia mentula, the aerobic intracellular rH was always between 19 and 22. Later, Chambers, Pollack & Cohen working with the sand-dollar egg {Echinarachnius parma) found a lower overall rH (about i2-o). We did no anaerobic experiments but the American workers got a value of 7-9 in hydrogen. No rhythmic changes occurred during segmentation nor was there any localisation of reducing power in the egg-cells. These observations were confirmed by Chambers, Pollack & Cohen. Cytolysis in amoebae produces an increased reducing power, but we were never able to see this in the case of the eggs, though we expected to do so in view of the statement made by Faure-Fremiet that on cytolysis the reducing power for methylene blue of a given amount of Sabellaria eggs rises some 30 to 40 times. Chambers, Pollack & Cohen found the exact opposite to be true in the case of the sand-dollar egg; i.e. it becomes less reducing as it cytolyses. We attached much importance to the fact that no change in intracellular rH occurred on fertilisation, for it implied that the intensity of oxidation-reduction was not affected by that event, and the tremendous increase in oxygen-consumption associated with it must therefore be a quantitative rather than a qualitative change^. The same substances are combusted, we concluded, before as after fertihsation, only in less quantity. This agrees with Runnstrom's view that fertilisation affects the degree of dispersion of the egg-colloids, and increases the accessibility of the enzyme surfaces for their appropriate substrates. We also found the rH to be quite constant as far as the 8-cell stage.
Cannan investigated the oxidation-reduction potential of echinochrome, the reversible natural rH indicator extracted from the eggs of Arbacia. This pigment takes a definite place on the rH scale, but does not form a dissociable compound with oxygen, so that its role in the cell must be an activator rather than a carrier of oxygen. The pigment may therefore be an effective oxygen activator in the cell, much as the " Atmungsferment " of Warburg is supposed to be. In this case, the concentration of its reduced form present would be of great importance, and this would be determined by the oxidationreduction potential of the cell or the phase in which the pigment is present. Arguing in this way, Cannan pointed out that only a very small change in intracellular rH might increase or decrease the metabolic rate or oxygen consumption by a hundredfold, so that our inability to find any change in rH of the cell at fertilisation — a time when the respiratory rate abruptly and greatly increases — involved no contradiction. In the hypothetical system pictured by Cannan, very wide metabolic latitude is combined with stability of rH, i.e. a poised oxidation-reduction potential, and this is what actually seems to be the state of affairs in the living cell.
- See further, on this subject, p. 626. Fertihsation involves " the opening of doors within the egg-cell".
Cannan pointed out that in the eggs of Arbacia echinochrome is in the fully oxidised state, but that in those of Echinus it is partially reduced. As the mid-point of its rH titration curve at pW 7 is about rH 6, it may be concluded that the cell-granules of the eggs of Arbacia are more oxidising than this, while those of Echinus esculentus eggs are at about that figure. From the work of Vies & VelHnger, then, we may conclude that these portions of the egg-cell have a rather acid />H, and from that of Cannan that they have a distinctly more reducing rH than the rest of the cell. Such a state of affairs may be usual in cells; thus mitochondria reduce Janus green, a very reducing dye (Needham & Needham), and Cannan found that hermidine, a natural indicator contained in plant cells, was held reduced there although active photosynthesis with oxygen production was going on, and although the micro-injection method in the hands of Rapkine & Wurmser, and other indicator work by Brooks, had demonstrated the overall rH of plant cells to be about 17. The living cell is without doubt extremely heterogeneous.
Micro-injection studies of intracellular rH were extended by Rapkine & Wurmser, who determined the average rH of nucleus and cytoplasm separately in the eggs of Strongylocentrotus lividus and Asterias rubens. There was absolutely no difference, both lying between rH 19 and 20-5. Thus the old idea of the nucleus as an "oxidation-place" (LiUie and Unna) is devoid of foundation. It remains possible, of course, that oxidations may go on to a greater extent in the nucleus than in the cytoplasm, though there is no evidence for this view; what is certain is that there is no greater intensity of oxidation-reduction in the nucleus than in the cytoplasm. They did not follow the eggs through later stages because of the disappearance of the germinal vesicle. Rapkine also studied the rH of the liquid fining the blastocoele cavity in echinoderms, micro-injecting indicators into it. He found it to be about 19. As he had strong reasons for supposing that free oxygen was circulating in it, it was clear that molecular oxygen was quite inactive as regards the oxidationreduction equiHbria. As the rH of this system was inferior to that corresponding to the equilibrium between hydrogen and oxygen in water vapour (rH 28), Rapkine concluded that the activation of oxygen in the sense of Warburg no less than the mobilisation of hydrogen, in the sense of Wieland, must be important here. Much other work on plant cells, where actual bubbles of molecular oxygen may be seen traversing a protoplasm of rH as low as 17, led to the same conclusions, and what Clark, Cannan and Cohen call the potential of "biological" oxygen must be much lower than that of the same gas in the molecular condition.
The colorimetric rH measurements of Needham & Needham on marine egg-cells were afterwards confirmed by Vellinger, using a direct electrode potential method on a thawing "puree" oi Strongplocentrotus eggs. The result so obtained, when corrected for temperature and dilution, came to rH 20, that of sea water to rH 26. The fact that our measurements and those of Vellinger corresponded so well as regards rH and so badly as regards pH. might be explained by the assumption that cytolysis does not affect the rH, but does the />H, in this material.
It then occurred to Reiss & VelUnger to ask whether developing sea-urchin eggs could get their energy from hydrogen-acceptors, and not from molecular oxygen, i.e. whether the eggs could carry on their metabolism if put under anaerobic conditions. Eggs were placed in anaerobic solutions poised at different points on the rH scale by indicators, haemoglobin and other substances, and it was found that if the potential was 200 mv. or over, i.e. rH 23 or above, the cleavages would go on as usual, but if it was below that point, the number of cells dividing fell off rapidly until at 150 mv., or rH 22, no cells would divide. They concluded that the "rH d'arret" was always a little above the rH of the cell-interior. The work was shortly afterwards confirmed to some degree by Rapkine, and is very important in view of possible phases of " anaerobiosis " in embryonic life^ (see pp. 700 and 742). The determination of the upper limit of rH for cell-division was subsequently made by Reiss, using potassium permanganate, sodium hypochlorite and ferricyanide. For sea-urchin eggs it was 665-720 mv. and for those of Sabellaria 600-700 mv.
1 There is no contradiction between these results and those of E. B. Harvey, who studied the effects of anaerobiosis on the cleavage of echinoderm eggs. In her experiments, the cells were merely stained with methylene blue as an indicator for the disappearance of oxygen and when this was reduced, cell-division ceased. In Rapkine's experiments an excess of reducible dye was present around the cells.
In 1929 Friedheim studied the rH of chick embryo Breis from different ages, using MichaeUs' mercury electrodes. There was no paralleHsm between rH and growth-promoting power (measured by the Carrel techniques), and the Breis seemed to be most reducing at the 13th day of development; thus:
in days
rH
Age in days
rH
5
6-7
13
3-9
7
4"9
15
5-^
9
4-6
17
8-6
II
4-3
19
lO-I
The only investigation of the rH of avian yolk and white is that of Pa\'lov & Issakova-Keo who used a platinum electrode. The yolk of the infertile unincubated egg was always some hundred millivolts more positive than the white: the average rH of the former was about 24, that of the latter 28. If the eggs were kept at room temperature the EA of the white showed a continual tendency to become more positive, reaching after a fortnight a level of + 0-42 or rH 32. If incubated at 37° this positive trend occurred also in the absence of development, but if the egg was fertile the white showed on the contrary a negative trend leading to EA + o-io at 1 1 days, or rH 20. The yolk followed a corresponding downward course, leading to EA + 0-30 at 1 1 days or rH 18: thus always keeping at a less reducing level than the white. These facts agree well with the well-known negative trend found in cell-suspensions or bacterial cultures and indicate that the developing embryo gives off reducing substances to the rest of the egg.
6-4. Water-metabolism of the Avian Egg
It will be convenient to speak next of the water metabolism of embryos, a subject of considerable importance. Although the bird's egg forms one of the most complicated of the systems we have to consider, it is yet one of the best understood, and for that reason it may be taken first, the discussion then passing on to the eggs of other organisms. Another reason for dealing with the hen's egg first is that in it the embryo can be separated from the yolk at a comparatively early stage, so that separate determinations of water in embryo and food-material can be done. This is much more difficult in many other cases, such as that of the frog.
In general we may say that from the earHest point examined the chick embryo loses water relatively, although its actual content of water in grams increases with its growth. This is shown by the curves in Fig. 220, taken from a number of investigators (Bialascewicz; Hasselbalch; Rubner; Iljin; Murray; Liebermann; Pott & Prey er) , which descend very regularly, following an S-shaped course from the beginning of incubation to the end. It is unfortunate that
Waber
content of chick embr^^o
B Tangl.
vTangI % v.Mibuch
Cahn
<$> Liebermann Bialascewicz © Murray
© Rubner
® lljin
Hasselbalch
^ Prevosb &, Morin
• Schmalhausen
D Romanov
I I I I I \ L
Days Hatching
I I I \ \ \ I I I I I I t I
9 10 1112 13 14 15 16 17 Fig. 220.
9 20 21 22
we have few data before the 5th day. A solitary determination of Bialascewicz indicates that the average value of 94 per cent., which we have at that time, may be a peak rather than a steady level and Schmalhausen, who gives a value of 70 per cent, for the second day, 85 per cent, for the third and 93 per cent, for the fourth, emphasizes this point. Further investigation is much needed here, but for the greater part of the incubation period the facts are very definite. The obvious corollary of this increasing dryness is that for every 100 gm. of water there will be more solids present at the end of development than at the beginning. The details of this concentration process were worked out for the chick embryo by Murray in the case of protein and fat, and by Needham in the case of total carbohydrate. As Fig. 221 shows, there is the gradual rise that would be expected a priori. It may be noticed that the only exception to this rule occurs in the case of the total carbohydrate, for from the 4th to the 8th day this value descends, showing the important part played by carbohydrate in the embryo in the \ery early stages. The protein uncorrected for that in the feathers has a peak on the 1 7th day, which is exactly what might be expected, since the feathers consist of practically dry protein, and it is about the 1 7th day that they form the highest percentage of the body-weight. The rise in the fat in relation to the water recalls the "lipocytic constant" and the other tissue constants suggested by Mayer & Schaeflferand Javillier & Allaire. Their behaviour during the development of the chick is very interesting (see Section 12-5). It will be important in the future to investigate further the lipoid and sterol content and concentration of the embryo, especially with a view to unravelling their influence on surface phenomena in development. The "Nachahmung" school of workers have already shown how likely it is that surface factors are one of the most important foundations of morphogenesis; it remains to establish by direct enquiry that this is really the case.
The second important factor in the water metabolism of the developing avian egg is the loss of water through the shell to the environment. At the opening of the Section on respiration a number of citations were made which showed that the loss of weight which occurs during the 3 weeks of incubation has been known for a long time, ever since the eighteenth century. EstabHshed by so many workers for the hen, Groebbels and Groebbels & Mobert have recently extended it to the eggs of many other birds, mostly wild species. Their data are fragmentary, and not suitable for compression, but in all cases they found that the larger the embryo, the less was its water-content. They also found a falling weight of the whole egg during development, four examples of which are given in Fig. 222. The amount of weight lost by eggs of various birds differed, but never exceeds 30 per cent of the original weight. Table loi gives these differences concisely. For a given bird, such as the hen, the loss is so constant that Zunz suggested frequent
Fig. 221,
Fig. 222.
weighing as a guide to regulation of normal development. It is interesting to recall that other terrestrial eggs, e.g. the silkworm (Luciani & Piutti) lose water as they develop.
Table 10 1. Loss of weight by fertile eggs during incubation.
Species
Hen ( Callus domesticus)
Hen( „ „ )
Hawk {Buteo buteo) ...
Falcon {Cerchneis tinnunculus)
Pheasant [Phasianus colchicus)
Nightingale {Turdus philomelos)
Yellow-hammer {Emberiza citrinella sylvestris)
Chaffinch {Fringilla coelebs)
Linnet {Acanthis cannabina)
Robin {Erithracus rubecula)
Sparrow {Prunella modularis) ...
Warbler {Sylvia communis) ... ...
Starling
N.B. This progressive water loss is one of the main difficulties in the way of successful in vitro incubation of the avian embryo, a technical problem which has not yet been solved; see Loisel (2 or 3 days), Vogelaar & Boogert (6 days), Fere; McWhorter & Whipple and S. Paton.
%
Investigator
17-9 14-9
1 1-5
Tangl
Murray
Groebbels & Mobert
23-0
II-8
26-8
12-2
IPs
1 7-0 12-55
Tangl
Iljin and Alcacid working with different incubators, some using wet air and others dry, observed a greater loss of weight in the latter, but the most complete examination of evaporation-rate is that of Murray. His immediate aim was to find the optimum conditions for development, and to lay these down as a standard environment for future work, but his investigation of the causes of loss of weight during incubation was exhaustive. The most accurate method of finding the surface of the egg, Murray found, was expressed by the equation S = 5-07 . W^ (cf Dunn & Schneider's S = 4-63 x W^), but when the surface so obtained was correlated with the weight loss at standard constant conditions of temperature and external humidity, there was no significant relation.
Murray next found the weight of the shell per square centimetre, and, correlating that with the weight loss, observed a much closer correspondence. The conclusion therefore was that thickness was a more important factor in determining water loss than area, but that the influence of both these factors was negligibly small. Probably an egg with a heavy shell which has small rarefied areas may lose more weight in a given time than one which has a lighter shell of uniform thickness. Eggs in which minute cracks were made supported this view by losing as much as 100 per cent, more weight each day than the average normal eggs. Murray weighed the shells taken from eggs of different incubation times, and comparing his results with those of Carpiaux; Tangl; and Plimmer & Lowndes, who had made shell weighings in connection with calcium analyses, he concluded that on the average o-oi gm. of shell-substance are lost to the interior of the egg for every increase in embryo weight of i -o gm.
He next studied the loss of water by the eggs at different positions of the two main variables, i.e. temperature and humidity. Fig. 223, taken from his paper, shows the loss in weight of White Leghorn hen's eggs during the incubation period in standard conditions: T ^ 38-8 ± 4°, humidity 67-5 ± 2-5 per cent., continuous flow of warm air, eggs turned once a day. There was no perceptible difference between fertile and infertile eggs until the i6th day was reached, after which the fertile ones tended to lose more weight than the infertile ones. In Fig. 224 is shown the effect of humidity; evidently the most important factor of all. At 100 per cent, humidity the egg loses no weight. With regard to the fact that the fertile eggs lost constantly rather more weight during the last week of incubation than the infertile ones (found also by Bywaters & Roue and Romanov), Murray pointed out that three possibilities presented themselves to account for this: (i) that the expired carbon dioxide was greater by weight than the oxygen absorbed during the same period, (2) that some other gaseous product was eliminated, or (3) that there was an increased evaporation of water. Neither of the first two of these theories is very agreeable with the facts, for, if ( I ) were true, the predominant substance combusted during the last week would have to be protein, and we know almost certainly that this is not the case, while all that is known about the metaboUsm of the egg is against the second possibility (see the Section on respiration). The third alternative must then be the correct one, and, although it is not easy to see how the presence of the developing embryo could increase the water elimination, it must be remembered that the embryo is producing heat, and that evaporation would be accelerated thereby. Probably the circulating blood in the allantoic membranes, said Murray, is a more efficient evaporating mechanism than the undifferentiated albumen.
Fig. 223.
The dependence of water loss on external humidity demonstrated by Murray shows that birds' eggs have no power of controlling the amount of water they lose. This conclusion had been previously arrived at by Aggazzotti, who studied the water loss from hen's eggs at sea level, and at the high-altitude research station at Col d'Olen. He found that at a height of 250 metres above sea level the eggs lost more water each day than at Turin (sea level), exactly contrary to what took place in the case of the adult animals, which lost more water each day at Turin than at Col d'Olen. There was evidently no regulatory mechanism in the hen's egg, and Aggazzotti compared the initial unprotected state of the egg as regards water loss with the assumption of homoiothermicity which takes place during development.
Fig. 224.
Interesting experiments on the evaporation rate of hen's eggs have been carried out by Dunn. Individual eggs of White Leghorn breed gave evidence of great variations in the rate at which they lost water. The data for the effect of incubation, presence of living embryo, etc.. were in general agreement with the later independent results of Murray. Dunn found that eggs were two or three times as variable in their evaporation-rate as in their original fresh weight. If, then, the rate at which an egg loses weight is regarded as an index of the permeability or porosity of its envelopes, then the shells of eggs must be more variable than the sizes of the eggs themselves. This was exactly what had been found by Curtis for the weights of the shell, the coefficients of variation of shell- weight/egg- weight being I0-43/6-36. In sum, loss of weight, said Dunn, was to be regarded as an individual character, like weight, length, breadth, shell-weight, yolk-weight, etc. In his second paper, he studied the relation of eggsize to weight loss, and concluded that larger eggs, though they lose more actual weight, lose an appreciably smaller proportion of their weight than smaller eggs. The larger eggs can apparently better conserve their moisture-content, but this is due wholly to their relative surface. The shells of the larger egg, however, were somewhat less porous, for they lost less weight per unit of surface area. This shell-difference was the subject of the third paper. Analyses made on the shells of eggs which had shown high and low rates of evaporation respectively revealed no difference except in the shell-weight, thus:
Egg- Shell- Shell- Calcium Magnesium
Rate weight weight weight oxide oxide
of loss (gm.) (gm.) (%) (%) (%)
High 65-22 5-399 8-28 52-01 1-52
Low 55-38 5-258 9-49 52-44 1-52
But, on the other hand, the rate of evaporation was correlated very closely with the number of pores (see Rizzo, p. 722) in the shell, and this was indeed quantitatively much the most important factor. Later Dunn investigated the relations between evaporation-rate and hatchability. He found that under constant conditions the rate at which a normal fertile egg loses weight in the first 7 days of incubation played little or no part in determining the subsequent fate of the embryo contained in it. Nor was the weight loss during the 2nd week correlated in any way with the hatchability. On the other hand Romanov found that the optimum hatch occurs at 60 per cent, humidity. He weighed a large number of embryos from eggs kept at 40 and 80 per cent, humidity, and concluded that the latter condition gave slightly heavier embryos than normal although their percentage dry weight was less than usual. At high humidity the percentage ash was high in the embryonic body. Too high humidity gave, he found, a worse mortahty than too low.
We have seen, then, that the egg as a whole is continually losing water at a constant rate, and that the embryo is continually gaining it at an ever-decreasing rate, so that it becomes less and less wet as it develops. The third cardinal fact in the water metabolism of the hen's, egg is that the yolk is gaining water at the expense of the white. This process has already been alluded to in the Section on biophysical phenomena, where the viscosity and the osmotic pressure of the yolk and the white necessitated its mention. There is reason, in fact, for believing that the yolk absorbs water from the white from the moment at which they first come into contact, i.e. in the oviduct, but it is probable that the mechanism by which this is done differs as time elapses, and as the embryo grows. The actual determinations of the water-content of yolk and white are assembled in Fig. 225, from which it can be seen that the results of all investigators from Prevost & Morin onwards agree well together. A little uncertainty, however, exists with regard to the course taken by the yolk after the mid-point of development, for Bellini's figures would
Fig. 225.
make it seem as if its water-content remains unaltered after that time, whereas the results of other workers show a descent to about the same value as in the unincubated egg. It is probable that there is a descent, for the dense and viscous condition of the yolk at hatching is familiar^. The course of affairs, then, maybe summed up by saying that for the first 10 days a flow of water passes from white to yolk. After that time the water-content of the white remains constant, but, owing to its now very small size, becomes quite unimportant in absolute reckoning, while that of the yolk declines again to its original figure. Carini, without giving any figures, stated that the yolkvolume increased at the expense of that of the white, which is in agreement with the rest of our knowledge about it. He also found that the digestibility of egg-white by pepsin decreased as incubation proceeded, and he put this down to its decreasing water-content.
What is the mechanism of this passage of water into the yolk? Greenlee supposed that the greater concentration of osmotically active substances in the yolk would amply account for it, and that the transference of water was entirely due to osmotic causes. He found that the rate of loss of water by infertile whites followed an extremely regular course, rising with the temperature, and falling with the time. He constructed curves for this, and was able to express the whole process by an equation on the basis of which the moisture content of the yolk and white of an infertile egg at any given time after laying could be predicted for a given temperature and a given initial state. But the evidence in favour of the water-current of infertile eggs being purely osmotic in nature is not satisfactory (see p. 816).
In the developing egg, however, matters are certainly more complicated. Vladimirov criticised the older viewpoint, showing by means of a simple calculation that the observed osmotic pressures would not account for the movements of the water. He suggested that an important factor besides osmotic pressure was the amount of water held back by the protein of the egg-white, the "Aufquellungswasser". In protein solutions of above 40 per cent, the intensity of this force can reach several atmospheres. In order to penetrate further into these complex relations, Vladimirov measured the electrical conductivity of the egg-white during development, as an index of what was happening to the electrolytes. The results are shown in Fig. 226. If the eggs were infertile and not incubated, there was no change, the electrical conductivity remaining in the close neighbourhood of 7-6 . 10^ Kohlrausch units (i K. unit = the conductivity of a substance of which a column
- The question of the " bound " water in the egg is one of much interest. Bound water may be defined as that part of the water in which added solutes will not dissolve. Zawadzki showed that the free water in the yolk is probably identical with the intermicellar liquid (the ultrafiltrate) of Bialascewicz (seep. 361). By adding known amounts of sucrose and urea Hill found that 97 % of the water in the egg-white was free, and 85 % of that in the yolk. One egg-yolk would thus contain (besides 7-95 gms. solid) 6-05 gms. of free and 1-05 gms. of bound water ; one egg-white would contain (besides 6-39 gms. solid) 22-9 gms. of free and 0-71 gms. of boimd water. Nothing is known of the variations which may occur in these factors during the development of the embryo, or of the bound water in the embryo itself.
Fig. 226.
I c.cm. long and i sq. cm. in area opposes a resistance of i ohm). If the eggs were infertile and yet were incubated, there were only small changes, which led to an increased conductivity, but if normal development went on, there was a marked downward trend, the conductivity reaching a minimum of 2-53 .10^ on the 20th day. These results are in agreement with those of Bellini (see above, p. 830), and would have indicated a definite decrease in the electrolytes present in the white had not Vladimirov made a correction for the large amount of protein present. This was based on theoretical grounds (see the original paper) and showed, as appears from the dotted line in Fig. 226, that the electrolyte-content really remains constant, i.e. the absorption of electrolytes moves parallel with the absorption of water, and can therefore play little or no part in the mechanism governing the latter phenomenon. Osmotic pressure measurements, which have already been referred to above, led Vladimirov to the same conclusion. After the 6th day, there is little or no change in the osmotic pressure of the egg-white. Finally, he examined the pH of the egg-white at different stages, with the results which have already been described (see Fig. 211). His pH measurements were done after removal of carbon dioxide by bubbling of hydrogen and subjection to a vacuum, so that the acid responsible for the decreasing pH of the white must have been a fixed one, and could not have been simply CO2 . Thus the tendency towards the acid side of neutrality, brought about by the presence of this unknown acid in increasing concentration, would lead, Vladimirov argued, to a closer approach to the isoelectric point of the egg-white proteins (between pH 5-0 and 6-o), and therefore to a loss of the power they possess of holding water, their "Quellungsfahigkeit". In this way the current of water yolkwards may be better explained. It should be noted that all the constituents of the white pass into the yolk or the embryo, but that when they do so at a much greater rate than the average we speak of a yolkwards current. Thus the salts also pass into the yolk, but not faster than the protein of the white is itself absorbed by the embryo.
Bartelmez & Riddle published some data which add to our knowledge of the current of water. The following table is striking :
Table 102.
%
water in yolk
'
Incubated
Incubated
Ov;
ary
Oviduct
Fresh-laid
24 hours
4-9 days
Gallus domesticus
Turtur orientalis
Streptopelia alba
Columbaoenas
Columba livia domesticus ...
46
17
46-79 55-21
47-59 55-83 55-80
54-71
48-40 56-94 57-11 56-80 57-17
58-80 58-68 59-93
Columba guinea
—
54-97
55-6i
60-15
They pointed out that, as far as the fowl was concerned, the time of most rapid absorption of water by the yolk was before laying, and they suggested that probably the liquid which fills the sub-germinal cavity in the bird's tgg was derived from this source. Digestion of the upper part of the latebra would play a part in the production of the sub-germinal cavity, as was first made likely by Patterson, and it is a remarkable coincidence, if no more, that the cavity is formed during the period of most rapid water absorption. Bartelmez
H Allanfcoic(Kamei) nAmniotic( ^ ) ® Allantoic (Rske&,Bqyden)
& Riddle found that the fluid filling the subgerminal cavity in the pigeon's egg would hardly form a coagulum when heated, and evidently contained only a little protein.
Closely associated with the water metabolism of the egg is the origin of the amniotic and allantoic liquids. Kamei has studied their increase in volume (see Fig. 227). The allantoic fluid shows at first a fairly regular rise and by the middle of development reaches
6 c.c, after which it probably falls. These 6 c.c. represent about 15 per cent, of all the water in the egg, i.e. that percentage has been required to assist in the excretion of from
7 mgm. (Fiske & Boyden) to 3I mgm. (Needham) of uric acid (see p. 1 092) . The reabsorption of water from the allantois must begin very soon after the mid-point of development, for by the 12th day its uric acid content is increasing while its volume is remaining steady or diminishing. The amniotic liquid, on the other hand, reaches what is practically its maximum by the loth day and does not fall until the final desiccation of the egg before hatching begins.
The specific gravity of the amniotic liquid rises to a maximum on the 14th day but that of the allantoic liquid rises throughout development, as follows :
Amniotic Allantoic
1-0062 1-0070
1-0630 1-0147 1-0400 1-0205
especially turtles, contain white^, and Agassiz gave a description of the histology of their yolk after laying, which has been interpreted by Bartelmez & Riddle as indicating
Fig. 227.
Day 9
17
The eggs of reptiles,
- Deraniyagala describes the white as at first viscid and later quite mobile and limpid. This is precisely the opposite to what happens in avian eggs (see Figs. 207 and 215) but would be expected from the considerable water-intake (see p. 898, Table 105).
an absorption of water by the yolk in that case also. Agassiz also noted a liquefaction of the albumen in the fowl's egg immediately over the germinal disc, corresponding to the liquefaction underneath it.
Fig. 228.
6-5. Water-content and Growth-rate
In birds, then, the general rule seems to be that the water-content of the embryos is higher the younger they are. In many other cases, the same relationship appears. Especially in the case of mammalian embryos is this true; thus Fig. 228, where a good deal of the data is collected, shows a continual fall for man (Fehling; Michel; and Brubacher), for the guinea-pig (Inaba), for the rabbit (Fehling), the mouse (von Bezold and Inaba), and the cow (Moulton, Trowbridge & Haigh). Moreover, the process is continued after birth, as the complete figures of Hatai for the rat, and of Thomas and of Weigert for the cat and dog clearly show. On the basis of these facts, several authors have concluded that a decreasing water-content is a universal accompaniment of growth, and the more rapidly the latter takes place the more rapidly does the drying up of the tissues go on. Cramer found that the watercontent of neoplasms was always higher by at least 5 per cent, than that of normal tissues, and that it varied directly with the growth-rate of the particular neoplasms, i.e. those growing the fastest as measured by mitotic index had the most water (see Fig. 229). Cramer concluded | that a high water-content | was intimately associated a with a high growth-rate. ■Then Ruzicka later was | much impressed by these facts, and enunciated a "law of protoplasmic hysteresis", while Rocasolano and Le
Fig. 229. 292, J, 63, T, 27 & 72 are strains and a and b generations of strains.
Breton & Schaeffer pointed out its relations with the paraplasmatisation of the tissues with age (see p. 747). Ruzicka regarded the ageing process as a continual tending of protoplasm towards stability, solidity, insolubility and dryness. Some curious digestion experiments which he made are relevant here. The time required to dissolve embryonic tissues by trypsin under standard conditions he found to vary as
follows : Hours
Frog. Blastulae ... ... ... ... 3-6
Embryos with tail buds ... ... 26-32
Embryos with external gills ... 43-53
Tadpoles 19 mm. long ... ... 71-80
In all cases there was an insoluble residue which increased in amount per gram of original material with age. Ruzicka also showed that the older an individual the more easily can flocculation of the proteins of its tissue juice be brought about. Thus the "Pressaft" of frog's eggs required 2-66 c.c. of 96 per cent, alcohol for flocculation, while that of lo-year old frogs required only 0-9 c.c. This phenomenon was related, he thought, to the isoelectric point of the cell-protein. Coherence, degree of condensation, hysteresis (passage from sol state to gel state) with many other concepts all intimately associated with
t: 70
SchaperO Org. subs. • Ash ® Water Davenport- Q Water
Bialascewics Water raure-Fremiet\<a Water &, Dragoiu
Williams © WaberRanaSilv. '♦ Bufo lenbig.
5 Years
decreasing water-content, form the components of Ruzicka's general theory.
In spite of this agreement, however, some facts had been known for a long time which seemed to oppose the introduction of a general law of decreasing water-content. The work of Davenport; Schaper; Faure-Fremiet & Dragoiu; and Bialascewicz on the early stages of amphibian development seems at first sight to be in contradiction with it, for, as Fig. 230 shows, the water-content rises steadily until about a week after hatching. Kaufmann, again, showed that salamander larvae cut out of the uterus took up much moisture when put into distilled water. Here, however, it must be remembered that the embryo cannot be separated from the yolk-sac, so it is the water-content of embryo plus yolk that is being measured. And where the yolk is allowed for, as in v. Bezold's work on Bombinator igneus, the water-content follows the usual rule and decreases, thus :
%
Embryo
90-6
Hatched larva
86-7
Tadpole
8l-2
Adult
77-3
On the other hand in the case of fish eggs it is possible to separate the embryo from the yolk from a comparatively early period onwards, and for the trout, where this has been done, the water-content of the embryonic body does not seem to change at all. Fig. 232, constructed from the figures of Kronfeld & Scheminzki and of Gray shows this very definitely, and it would seem that the water-content of the trout embryo never varies much from 85 per cent, though, of course, it may be higher in the earlier period which was not investigated. From the work of Faure-Fremiet on the egg of Sabellaria alveolata it is not possible to learn much, for the yolk was never separated from the embryo, and we cannot say therefore what was the significance of the loss of 5 per cent, in dry weight. Teissier's study of the development of the medusa Chrysaora hyoscella, however, demonstrated that, although the adult medusae have a great deal of water in their bodies, the early stages have much less. Thus he obtained the following results :
Table 103.
Organic Phos Ash substance phorus
2-3 31-4 0-33
3-2 25-8 0-27
But it might perhaps reasonably be argued that the water-content of medusae is so unusual that the ordinary relations would not be expected. Histological arguments supported the view that, while the early increase in water-content was due to intracellular swelling, the later increase was intercellular, due to the mesoglia.
Water per
gram dry
Age of medusae
% water
weight
/'<iday Planulae -' '"? ^^^^
64-9
1-84
66-3
1-97
2 j^y^ 1 3-4 days
67-5
2-07
71-0
2-45
Schyphistomes
94-5
17-00
Adult medusae
96-3
26-00
SECT. 6] OF THE EMBRYO 887
Wetzel's comparative work introduces further complications. He analysed the eggs of a number of different kinds of animals, and then adopted the unsatisfactory expedient of comparing the results with figures for adults of the same kind, taken from other investigations. Thus he drew up the following table :
Table 104.
Water-content %
Egg
Adult
(Wetzel)
(Sempolovski)
Sea-urchin (Strongylocentrotus lividus)
•" 77-9
—
Starfish [Asterias glacialis)
67-36
S>Tpid&r-cxah {Maia sqiiinada)
sm
Cray-fish {Astacus fluviatilis)
77-11
Crab {Cancer pagurus) ... '
—
62-64
Dogfish {Scyllium canicula)
43-6
—
Ray {Raia radiata)
80-67
But we cannot conclude from this table that the echinoderms get drier as they develop or that the Crustacea and elasmobranchs get wetter, for we learn nothing from it about what is the real centre of interest, the embryonic body itself. According to Ephrussi & Rapkine the sea-urchin's egg absorbs water from the sea.
Wet weight Dry weight Change: unfertilised egg as unity (- J-;; ;;; t^iS +9;o
Just as there is still much uncertainty about the behaviour of water in the entire embryonic organism, so the data we have for its separate tissues are rather complicated, if not contradictory. For muscle tissue, Jacubovitsch stated in 1893 that the water-content decreased with the age of the embryo; his figures are shown plotted in Fig, 231 together with those of Mendel & Leavenworth on the brain and liver of the pig. Bischov, again, had found the figures:
Whole man
Muscle
Newborn
66-4
81-7
33 years
58-5
75-67
Then Faure-Fremiet & Dragoiu, in their work on the embryonic lung in the sheep, obtained a curve which fell from the 7th to the 14th week, after which the determinations became impossible owing to the swallowed amniotic liquid. As regards nervous tissue, Glaser's results on the brains and spinal cords of Amblystoma indicated a constancy in water-content; thus the just-hatched Amblystoma brain
GENERAL METABOLISM
[PT. Ill
had 82-8 per cent, and the cord 79-0 per cent., while Donaldson found for Rana adult brain 84-9 per cent, and cord 80-5 per cent. Rosenheim, on the other hand, found in human brain 90-29 per cent, for the 36 weeks' foetus and 85-8 per cent, for the infant shortly after birth, and a trend in the same direction was found by Gundobin and by Koch, who obtained the following figures :
Brain of foetus of pig 50 mm. long Brain of foetus of pig 100 mm. long Brain of newly-born albino rat ... Brain of adult albino rat
0/ /o
90-78
91-01
89-58
78-10
It is safe to say, then, that, though in certain cases the watercontent of embryonic tissues seems to remain unchanged with age, there are a few instances of a rise, and the majority of experiments show that it falls. The explanation of this fall is difficult, though, as we have observed, some investigators have seen in it perhaps the most fundamental attribute of the ageing-process. A point which does not seem to have been noticed so far is that the high water-content in the early stages may be related to the importance of primitive connective tissue and its contained lymph. I have already drawn attention to the primitive undifferentiated connective tissue, first discovered by von
Szily, in connection with the initially high concentration of glucose in combination with protein^, and it will again be mentioned in connection with the relatively high silicon content of young embryos. But what is important here is the fact that connective tissue has always a high water-content, and this might explain the phenomenon now under discussion. Thus Skelton found that bleeding in mammals produced a flow of water from the tissues into the blood, and by appropriate experiments he was able to ascertain what per ^ See p. 566.
o Cowmuscle(jacubovitsch) ® Pig brain (Mendel S^ Leavenworth) © Pig liver ( " " " )
o Sheep lung(Faure-Fremieb«tDragoiu)
• Cow brain (Schlossberger) ' ■ " heart (
♦ " lungs ( A " muscle! ▼ » liver ( © " blood (
Cow length in cms.
■^
Cow&,Sheep .5 weeks Pig mm. 50
10 100
15 150
20 200
Fig. 231
SECT. 6] OF THE EMBRYO 889
centage had been contributed by each of the tissues. His figures were striking.
%
of the total water
contributed to the blood
Muscles
lo-o
Liver
8-0
Intestines
2-0
Spleen ...
0-3
Connective tissue
78-0
There is thus the possibility that the decreasing water-content of embryos may be simply an index of the decreasing amount of primitive connective tissue. From the point of view of paraplasmatisation or degree of diminution of active protoplasm, it would be desirable to test the truth of this view in some way. The original suggestion of connective tissue and lymph as playing this role is due to Schlossberger. From the experiments of Wiener we know that, in the human foetus, the lymph circulation is active from an early date. Foetal lymph has been analysed by Raske (see Section 23-7).
6-6. Water-absorption and the Evolution of the Terrestrial Egg
We may now return to the curve for the rising water-content of the amphibian larva (embryo plus yolk) established by so many workers and plotted in Fig. 230. It has usually been regarded as a fundamental property of this phase of embryonic growth, but Arager contests such a view. Arager removed the jellies from frog neurulae, and put them to develop in a chamber the humidity of which could be controlled. In this way it was possible to produce larvae of very much less water-content than normal, and Arager noted that, in such larvae, the histological differentiation of the tissues was normal, although the morphological relations of the organs might be considerably upset.
But leaving this difficult question, we shall find it advantageous to study the results obtained by Kronfeld & Scheminzki and by Gray on the trout embryo. As has before been mentioned, this can be separated from its yolk at an early period. In general, it resembles the amphibia, for a preliminary period inside the egg-membranes is succeeded by a free-swimming period in which the fish lives on the yolk in its yolk-sac, and this in turn by a period in which ordinary ingestion of food by the mouth is begun if there is anything present to eat. As Fig. 232 shows, the water-content of the trout embryo is
Sgo
GENERAL METABOLISM
constant at about 85 per cent, during the last third of the first period and the whole of the second period. The water-content of the yolk is again uniformly low, about 60 per cent., and the fact that the water-content of the whole system, embryo plus yolk, rises, is evidently due to the fact that the embryo is growing all the time in size relatively to the yolk which is diminishing in size. Where the water of the embryo comes from is a matter which we may shelve for the moment. Now there is a strong probability that the type of graph shown in Fig. 232 for the trout may also turn out to be applicable to the frog. Fig. 233 taken from Gray's paper, shows the decreasing
< egg K..S.
^— yt,|k-sac
^ -starvation
1
o-'^aU
>P-Jl=-^
=i— ,
oY"^
m5S?5-^^~
i^^
%
N/
1^
1 ^Level oi'adultfish(Gray,Pearseetc)
Amphibia Bombinator igneue Von Bezold 11 Rana fcemporaria Glaser
\rtm
i^
■^
Amblystoma punctatum
^^^^
Pisces Salmo fario TanglS^Farkas Kronfeld«.Scheminz
'^"?;5
sT® — 5)__®—
-® "
. - „ .. Gray
Pearse
1 1 1 1
-J
Days
" i Perca-flavescena Pearse 1 1 1 1 1
Embryo <3>
Embryo O
Embryo+ Yolk*
Yolk alone ®
Embryo O
Embryo+Yolk*
Embryo + YolkJ
Fig. 232.
dry weight content of the larva (embryo plus yolk) in the frog and the trout, so that, as far as that goes, they behave similarly. It was said above that the frog larva is not capable of being separated into its embryonic and yolk components, but this is not strictly true, for a few careful dissections made by Glaser are available to throw light on the matter. Glaser separated the embryos of Rana temporaria into two parts, one predominantly consisting of yolk, the other of "nervous tissue". Water estimations on these gave the figure of 54-2 per cent, for the yolk and 8o-i per cent, for the embryonic body; these are plotted on Fig. 232, from which it can be seen that the relations are just like those found by the workers on the trout. It is, therefore, probably right to conclude that what happens both in the frog and the trout is a constancy in water-content of the em
SECT. 6]
OF THE EMBRYO
bryonic body, and a rise in the water-content of the embryo-plusyolk system.
The obvious question now arises, what is the origin of the water which helps to build up the tissues of the frog and the trout embryo. Gray's work on the brown trout, Salmofario, provides a graph showing the relative amount of embryo and yolk in the different stages ; this is given in Fig. 234. The wet weight of the embryo in percentage of
50r
oTrout
60 70 80 90
Days after hatchinci
Diagram illusbrabing change in water content of Larvae Fig- 233.
the wet weight of the system embryo plus yolk is plotted agains the time, and the resulting curve is S-shaped. It contributes to the view that the rising water-content of the whole system (shown in Fig. 235, which is also roughly S-shaped), is due entirely to the increasing preponderance of embryonic tissues, which maintain a constant proportion of water within themselves. "In other words", as Gray says, "as yolk is converted into embryo, water is added from the external environment and the yolk may be regarded as more or less desiccated nutriment. We can therefore conclude that the concepts of ' passive ' and ' active ' growth (Davenport) have no
892
GENERAL METABOLISM
[pT. m
foundation in fact." Now the initial weight of wet yolk in a newly fertilised trout's egg is approximately loo mgm., and, as 41 mgm. of dry yolk is present, an amount of fish can be built up with these materials corresponding to 256 mgm., for the water-content of the
100
^
90
_
/^
«
\ /^
I,
/ •
-^80
/
s.
f*
a
1
•I7O
_
'/
^
Before Hatching
After / Hatching
"«^
i
r
JO 60
1
Si
J
f
•^
1
|50
_
1
J
X
1
5
1
ki 40
1
"^
k
.*i
I
1 30
r
•0
J
i
l
20
/
10
1 1 1 1 1
20
70
80
90
30 40 50 60
Days after Fertilisation
Fig. 234.
final product may be taken at 84 per cent. Thus there is enough dry material in the fertilised egg to produce an amount of fish two and a half times its own weight. But, on the other hand, there are only 59 mgm. of water present in the original yolk, and this will be sufficient for only 70 mgm. wet weight of fish. And the observed weight of the fish at the end of the yolk conversion phase of development is actually about 150 mgm. The explanation of all this is, of course, that some of the initial dry material is lost during develop
SECT. 6]
OF THE EMBRYO
893
ment by combustion, and a good deal of the eventual water is taken in from outside. Now between a quarter and a third of the initial dry material is so used, so that the eventual dry weight in the finished fish is about 25 mgm., which at 84 per cent, water needs 155 — 25, i.e. 130 mgm. water. But, as there were only 59 mgm. water present in the yolk at the beginning, 130 — 59, i.e. 71 mgm., must have been absorbed from the exterior. These relationships are shown in the chart, constructed by Gray and given in Fig. 236. It will be seen at a
45
10 20 30 40 50 60 70 80 Days after Fertilisation Fig. 235.
90 100 no
glance that the newly fertilised tgg contains enough solid for constructing the finished embryo and for providing the material for combustion to serve the basal metabolic requirements, but it does not contain even half enough water. The latter has, therefore, to be taken from the aqueous environment, according to the following generalised equation : +
Wet
yolk
(i-ogm.
External
water (0-7 gm.)
Wet
fish
(1-56 gm.)
Dry yolk used for
other purposes
(o-i4gm.)
By respiration experiments, as already remarked. Gray was able to account for all the yolk disappearing in the last of these fractions.
In a later paper Gray found that a peak exists in the wet weight of the larva (yolk plus embryo); this is illustrated in Fig. 237. Up to the 85th day the wet weight of the larva increases, but after that time it falls, although the wet weight of the embryo steadily increases throughout the period. Thus after the 85th day the yolk is
894
GENERAL METABOLISM
[PT. Ill
being used by the embryo faster than it can absorb water from outside, and this begins when about i-io gm. of yolk is left unconsumed. This is important with regard to growth-rate (see p. 406).
Diagram illustrablng the olevelopemenb of the ecjg of S.fario A.Newlj ferbilised eo|C) B. Larva 80 days old J read_y bo absorb foodb_yc)ub.
sac
\ Embryo
Fig. 236.
Ranzi's parallel work on the egg of the squid, Sepia officinalis, led to an equation similar to that given above for the trout:
Wet + External + External = Wet + Dry yolk used for yolk water ash squid other purposes
(i-ogm.) (o-ySgm.) (o-033gm.) (1-727 gm.) (0-089 gm.)
He obtained curves identical in general form with that shown in
SECT. 6]
OF THE EMBRYO
895
Figs, 232 and 235, the water-content of the whole system rising, that of the yolk remaining steady and that of the embryo slightly falling (82 to 75 per cent.). The total weight of this egg rises considerably, from 76-9 to 132-8 mgm. and as will be shown later, ash as well as water is absorbed during development. The undeveloped egg of
15-0
14-0
13-0
12-0
10-0
100
Sepia contains 40-4 mgm. of water but the hatched embryo ioo-6 mgm. so that 60 per cent, of the final amount must have been taken in from the sea. The percentage of water in the system correspondingly rose from 52-5 to 75-81.
The work of Weismann indicated a similar absorption of water during the embryogeny of the cladoceran, Daphnia, and this was con 1 An equation for the egg of the axolotl, Amblystoma, can be derived from Dempster's data :
Wet + External = Wet + Dry yolk used for
yolk water axolotl other purposes
(i-ogm.) (3-92 gm.) (4-84 gm.) (0086 gm.) Ranzi's figures apply to the pre-hatching period only, Dempster's apply to the whole yolk-sac period as well.
896 GENERAL METABOLISM [pt. iii
clusively demonstrated by Ramult for Daphnia, Ceriodaphnia, Scapholeberis, and Simocephalus .
Another case in which it has been shown that the tgg contains enough solid but not enough water for the finished embryo is that of the ovoviviparous batoid elasmobranch, Torpedo marmorata. Davy found as long ago as 1834 that the mean weight of the egg when undeveloped was 182 grains, that of the egg plus early embryo was 1 77 grains, while that of the finished embryo was 479 grains. Allowing 80 per cent, of water for the mature state, which is very reasonable, only 95 grains would be required of non-combusted solid, so that it is very probable the increase was all due to water, especially as the gelatine-Hke egg-cases would hardly allow anything. but water to pass through them, Vidakovich afterwards found an increase in weight of the finished embryo over the egg, of 40 per cent., and from my own observations of the fish on which he worked, Squalus acanthias, I believe that the events which take place there are quite analogous to those in the trout, thus;
IV. tiV^Ull,, Ciil^O.
Water
Solids
(gm.)
(gm.)
Undeveloped egg
14-2
8-8
Finished embryo
35-0
6-0
Parker & Liversidge, again, reported that the undeveloped eggs of Mustelus antarticus, an ovoviviparous selachian, measured 43 x 16 X 10 mm. (roughly) while the ripe embryos ready to hatch measured 220 X 25 X 25 mm.
A very remarkable case is that of the (Siluroid) catfishes which incubate their embryos in their mouths. Wyman in 1857 studied several species of the genus Bagrus at Paramaribo in Dutch Guiana, and found that the hatched embryos not yet liberated from the parental mouth weighed considerably more than the undeveloped eggs. His conclusion that a nutritive fluid was supplied does not of necessity follow ; it is likely that a good deal of water was absorbed.
Again, in gastropods the eggs swell greatly, according to Nekrassov. As regards Crustacea Needham & Needham, in the course of other work on the chemical embryology of the sand-crab, Emerita analoga, observed that the water-content of the whole egg was 63 per cent, in the cleavage stages, 75 per cent, about the middle of development, and 85 per cent, at the time of hatching. But the extreme case is undoubtedly the desiccation that phyllopod (e.g. Artemia salina) and cladoceran eggs may go through before their development. Wolf found
SECT. 6] OF THE EMBRYO 897
that phyllopod eggs would withstand 14 years' drying in a desiccator without losing their power of development and Pirie has exposed Artemia eggs to phosphorus pentoxide at less than | mm. pressure for several days after which development proceeded normally. Carpenter noted much the same facts in the case of rotifer eggs. When they are put into water they develop : all the water in the body of the embryo being derived from the environment except that small trace which is bound to the hydrophihc colloids of the ^gg (see Gortner & Newton, and Robinson). Doubtless this hardiness is an adaptation to the seasonal drying up of the pools in which these animals live.
There is strong reason to believe, therefore, that the eggs of aquatic animals do not contain enough water to make their end products, but have to absorb it from their surroundings. It is possible that some obscure phenomena may receive an explanation on these grounds; thus the eggs of the pike (Esox), according to Kasanski, rotate round and round within their membranes, after only 24 hours cleavage; and this movement, which continues until the muscles are formed, may be an adaptation for ensuring water-intake, by setting up currents within the egg-case. And Amemiya states that the eggs of the fresh-water teleost, Oryzias talipes, show conspicuous undulating movements of the blastoderm from an extremely early stage onwards. Giard, long ago, showed that many aquatic eggs (fresh-water molluscs, Hirudinea, marine polychaetes, molluscs and nudibranchs) would develop well if kept in moist trays, not actually immersed in water, but that moisture was essential. Certainly in many cases, as we have seen, the percentage dry weight of aquatic eggs is much higher than that of the corresponding fully formed tissues.
"If then", said Gray, "it may be assumed that most, if not all, aquatic organisms are dependent on the environment for a supply of water, an interesting problem of phylogeny is opened up. The primitive vertebrate is, with good reason, regarded as the offspring of an aquatic type, but at some stage in the history of the truly terrestrial animals there must have come a time when oviposition occurred on land and not in water. During the earlier stages of their evolution terrestrial vertebrates no doubt laid their eggs in water where possibly the factor of greatest survival value consisted in the newly hatched individual having reached a stage at which it might fend for itself and be of active habit. Now an animal such as the trout necessarily hatches at an early stage since the increasing volume
898 GENERAL METABOLISM [pt. iii
of the lana soon reaches the volume of the original egg-shell ; further development without hatching would crush the embryo against the egg-membranes. In aquatic animals a postponement in the date of hatching is only possible when either the original egg-membrane is highly elastic or when it is separated from the yolk by a wide perivitelline space. Now an aquatic egg containing sufficient perivitelline space to allow the act of hatching to be postponed until the young organism is fully formed (and comparatively unhampered by remaining yolk) would form just the system most easily adaptable to terrestrial habits. Such an egg laid on land would undergo its normal development as long as it was protected from undue evaporation during the incubation period. Thus the experimental data we have point strongly to the suggestion that the Reptilia and their derivatives arose from a type of organism whose eggs were laid in water but which did not hatch until the yolk-sac period of development had reached an advanced stage. The wide perivitelline space which was then a necessary feature for the accommodation of the enlarging embryo became on land the means whereby water was supplied for development. The significance of the white of the hen's egg can be realised by the fact that after 19 days of incubation the embryo has absorbed i o cc. of water from it. The eggs of all birds and of many reptiles are of the same type, but in some reptiles the necessary water is partially supplied from external sources, and the eggs swell appreciably after being laid" (e.g. Sphenodon — Dendy; Dermochelys — • Deraniyagala; and many others).
A remarkable illustration of this is suppliedby the workof Karashima on the Japanese marine turtle, Thalassochelys corticata, which lays eggs the size of pingpong balls in the damp sand above high-water mark. He did not himself calculate the movements of water in these eggs but this can easily be done from his figures, with the following result :
Table
105.
Water in gt
■ams per 100
eggs.
Days of development
White
Yolk
Embryo
Allantoic
and amniotic
liquids
Total
Absorption
1330
1381
—
—
2711
87
78
971
15
699
2096
3
2798
30
520
1783
131
442
2876
Hatched
225
1262 1655
2360
3847
SECT. 6] OF THE EMBRYO 899
This table demonstrates {a) that the chelonian yolk absorbs water from the white, just as does that of the bird — a process which will tend to dilute the yolk appreciably if it goes on faster than the formation of embryo and amniotic liquid; and {b) that 1136 gm. of water are absorbed from the exterior by 100 eggs, i.e. 42-0 per cent, of the original amount provided by the maternal organism, which thus expects its embryos to obtain for themselves about a third of the water they require. A swelling of the egg must certainly have taken place, though Karashima did not report it : and this lack of water may in part account for the relatively small size of many chelonian eggs.
Still more remarkable is the fact, reported by Cunningham, that another turtle (from North Carolina), Chrysemys cinerea, has a special mechanism for wetting the earth in which the eggs are to incubate. "These turtles", says Cunningham, "select high ground in which to build their nests, sometimes a considerable distance from water. Usually the ground chosen is hard and dry, but a sandy beach may be used. The dirt is first moistened by water from a supernumerary bladder; as is shown by the fact that the dirt in the hole and surrounding it is wet while that further away is hard and dry." Cunningham analysed the water in this bladder (the function of which had previously been unknown) and found it to be a dilute urine, containing only 0-0005 P^r cent, of nitrogen. During development the eggs swell somewhat. This is a notable link in the evolutionary chain, for here the turtle goes out of its way to provide a store of water for its terrestrial eggs, yet outside not inside them. This must be the furthest point to which a non-cleidoic egg could go in a terrestrial environment (see p. 1 103). Moulton states that terrapin eggs have been commercially incubated by putting them in sand and sprinkling the surface every week until hatching, and Hochstetter reports success by a similar method on Emys europaea. Deraniyagala and Hildebrand & Hatsel find that loggerhead turtle's eggs {Caretta) take up a good deal of water during incubation. "Apart from other advantages". Gray continued " the value of the invention of viviparity is obvious; it entirely removes the necessity for the parent organism to provide in the newly fertilised egg enough water to last the embryo throughout the whole developmental period. Now it is known that the vitelline membrane in the hen's egg is permeable to water but not to salts and other osmotically active substances. Were the yolk surrounded
900 GENERAL METABOLISM [pt. iii
by pure water, the latter would rapidly pass into the yolk and thereby produce a large and mechanically weak ovum. If the aqueous surroundings, on the other hand, consisted of crystalloid substances the ease with which the embryo could obtain water would become increasingly less with increasing age owing to the rising osmotic concentration of the external salts. Since, however, the initial osmotic equilibrium between embryo and yolk on the one hand, and the aqueous surroundings on the other, is effected by means of a colloid, then although water is withdrawn from the latter its osmotic pressure does not rise as much as would that of a solution of a crystalloid. The existence of an albuminous solution round the yolk of terrestrially developing eggs appears to be an admirably adapted mechanism for providing the growing embryo with water." Thus Gray's theory amounts to this, that we may see in the eggwhite of the hen's egg a mechanism for supplying the embryo with a relatively constant pressure-head of water. If the chick embryo were dependent on the yolk alone, it would never be able to construct its tissues, for the yolk has only about 45 per cent, of water and the chick about 80 per cent, at the time of hatching. It is hardly necessary to indicate the way in which the findings of Vladimirov, referred to above (p. 880), fit in with the general viewpoint of Gray, and it looks very much as if the acid which seems to be produced by the embryo and which appears in the white, is the regulatory or control device governing the transfer of water from egg-white to embryo.
Table 106 gives a succinct survey of the movements of water in the hen's egg; it was calculated by Gray from the experimental data of Murray. It clearly appears that at least two-thirds of the water in the finished chick embryo is derived from the albumen.
In a later paper Gray continued his exposition of the relations between the water metaboHsm of the embryo and the evolution of terrestrial vertebrates. Agreeing with Watson that the main problem of evolutionary modifications centres round the possibility of deri\ing one morphological type from another without requiring any functional discontinuity of the organs involved, he considered the origin of the egg-white in birds. No reptilian or avian egg exists without an albuminous phase, and we may assume that its function is not radically different from that of the egg-white of the chick. The amniota, then, solved the problem of providing their embryos with
SECT. 6] OF THE EMBRYO 901
an adequate water-supply by coating their eggs with a watery protein-containing mass. "Now it is hardly conceivable", said Gray, "that the albuminous layer of the amnio te egg arose de novo as an adaptation to terrestrial life, for this would involve a sudden change in the structure of the oviduct. By the principle of
Table 106.
Survey of the movements of water in the hen's egg. Water in grams
& 2 Ji%^ ^%r
II I 1 111 %tl Pt
qI £ £ ill ►Sffs c^'s'
o-o 8-5 29-9 o-o o-o
o-oi
\t
0-4 8-45 27-2 2-4
i-i 8-4 25-4 3-5 0-05
2-5 8-2 23-0 4-6 0-I2
4-6 7-8 20-4 5-6 0-27
7'9 6-9 16-9 6-7 0-50
I2-0 5-3 12-6 7-8 o-8o
18 i8-i 2-3 9-2 8-8 1-20
20 27-4 I-O 2-2 9-8 2-00
7-5 27-7 Lost from yolk and white respectively
35-2 Lost from both
— — — 35-2
9-8
25-4 Lost other than by evaporation +2-00 by synthesis =27-4 of which, even if the whole of the water of the yolk went to form the embryonic tissues (which is unlikely) 20-0 gm. or 73 per cent, would be water passing from eggwhite to embryo. Compare this Table with Table 1 05 ; whereas the avian egg loses some 25-5 per cent, of its initial store the chelonian egg gains some 40-0 per cent, from the exterior. For the question of bound water, see p. 879.
physiological continuity it is much more reasonable to suppose this layer as equivalent to such homologous structures as are found in the anamniota. Among fishes tertiary egg-membranes rich in water are found in the Dipnoi, and they appear to be present in all amphibia. These membranes apparently protect the tgg against destruction by predatory animals though they may have subsidiary functions associated with the incubation of the embryo. In most amphibia, where the tertiary envelope is of a mucoid nature, the full
902 GENERAL METABOLISM [pt. iii
water-content of the envelope is not attained until after the egg has been deposited in water, but interesting and suggestive modifications are found in the eggs of those amphibia which deposit their eggs on land. In Phyllomedusa and in Rhacophorus the protective function of the mucoid envelope is to a large extent replaced by other devices, and it is difficult to resist the conclusion that the envelopes themselves are largely devoted to the provision of water. In Phyllomedusa hypochondrialis the eggs are deposited in the folds of leaves. The mucilaginous egg-capsules rapidly Hquefy after oviposition and provide a fluid medium in which the eggs develop. Agar observed that a certain percentage of capsules contain no eggs, and this suggests that the function of these membranes is to augment the amount of water available for the larva. The essential point is that the whole of the water necessary for development is provided by the walls of the maternal oviduct. Similarly the eggs of Rhacophorus schlegelii are laid in a subterranean burrow. Having formed this burrow, the female secretes into it a mucilage which with the aid of her feet is rapidly worked into a froth. Into this froth the eggs are laid and as development proceeds the froth is gradually liquefied. Here again all the water for development is derived from the female organs. From these types it is not difficult to derive either the egg of a reptile with its solid albumen phase or the egg of a bird with its fluid albumen which has entirely lost the power of protecting the embryo against predatory foes. It is interesting that far from requiring a supply of water from external sources, the eggs of birds fail to develop unless a certain amount of water is lost by evaporation during incubation, as Chattock has shown. And a suggestive experiment of Weldon's, who incubated eggs in such a way as to replace the amount of water normally lost by evaporation, indicates that the proper formation of the amnion is dependent on loss of water by evaporation.
"Since the mammals are derived from the oviparous reptiles it is of interest to consider how the small eutherian egg can be derived from that of the latter group without any break in the physiological functions of the organs concerned. A conceivable line of origin is suggested by the eggs of monotremes. These have no true albumen layer and the yolk ovum lies close under the shell. As it leaves the ovary, the egg is about 2 mm. in diameter, but during its passage down the oviduct its bulk is enormously increased so that the yolk is about 14 mm. in diameter before the shell is deposited (Caldwell).
SECT. 6] OF THE EMBRYO 903
This 300-fold increase in volume must largely be due to absorption of water, though a certain increase in dry weight may well occur. The only significant difference between an egg of a monotreme and an egg of a reptile is that, in the former, the aqueous secretions of the walls of the oviduct are passed straight into the yolky ovum itself instead of being deposited on its surface as a separate phase. In eutherian mammals this process has gone one step further since the water contained in the mother's blood is passed, not into the ovum, but direct into the embryo.
"If these arguments are sound", Gray continued, "there seems good evidence to show that terrestrial vertebrates have descended from a fish-like ancestor which possessed a glandular oviduct. The secretions of these oviducts were at first utilised as a protective covering to the eggs, but eventually they made it possible for the eggs to develop on land by providing an adequate supply of water to the embryo."
Gray's contention that there exists an evolutionary continuity between the amphibian egg-jelly and the avian egg-white, that they are, in fact, homologous structures, acquires still further interest from an isolated observation reported by Banta & Gortner, In the course of their work with Amblystoma, they found one day a mass of eggs contained in an opaque milky white jelly instead of the usual transparent and translucent material. Investigation showed that the dry weight of the normal jelly was 337 mgm. per cent, and that of the milky white one 361 mgm. per cent. When desiccated the jellies were indistinguishable in appearance but swelled up again to their original condition. The phenomenon was not, as far as could be ascertained, due to bacteria, and as the milky jelly has 9-18 per cent, nitrogen (dry weight) as against the 8-32 per cent, of the normal kind, they thought that perhaps the former might consist mainly of albumen instead of mucin. This was confirmed by qualitative tests. Here then was an aberrant example of an amphibian egg-jelly resembling the avian and reptilian egg-white, and indeed only requiring a shell to be transformed into it^. It affords an interesting commentary on Gray's remarks. The suggestion of Steudel & Osato mentioned on p. 331 may also be recalled — they pointed out that mucoprotein is not absent from the egg-white even of birds, and expressly referred to an evolutionaiy continuity with amphibia. The land-frogs offer some
1 Shells of a rudimentary kind do occur in amphibia (e.g. the land-frog Rana opisthodon and the African toad Xenopus laevis (Bles) ) .
N E II 58
904 GENERAL METABOLISM [pt. iii
attractive material for the study of these questions, e.g. the Eleutherodactylus of Dominica described by Howes, which lays large transparent crystal-clear eggs and has no external tadpole stage.
It is not without interest that these watery mucilaginous envelopes are found elsewhere in the animal world, e.g. in the eggs of certain insects, mostly Trichoptera (caddis-flies) and midges of the Chironomus class^. The nature of the protective function served by these jellies raises problems of much interest. I have already suggested that the reason why bacteria attack the amphibian egg-jelly so slowly is that it is practically a pure protein, mucin, and that a certain proportion of protein breakdown products is essential for good bacterial growth. There is also much evidence that similar properties may be ascribed to the avian egg-white. There is a large literature on the dietetic aspect of raw and cooked white of tgg which may be found summarised in the work of Bateman but without going into it at this point, it may be remarked that raw egg-white is very resistant to digestive enzymes, containing a definite antitrypsin and an antipepsin. In Section 19-3, moreover, we shall see that a natural bacteriolysin is present in raw egg-white (see Sharp & Whitaker) .
Insect eggs, indeed, provide further light on the evolution of terrestrial embryos; thus, work on the silkworm [Bombyx mori) brought out the following figures :
Farkas
Tichomirov
Water
Water
content
content
64-56
64-49
71-78
69-80
13-98
—
the finished 1;
arva has a
Httle
Unincubated eggs
Finished larvae
Unused material, membranes, etc. ...
In the case of this insect, at any rate, more water in it than the original tgg, but the explanation may lie in the fact that at hatching a notable quantity of very dry material is left behind. During metamorphosis also, the organism seems to become richer in water, but here again a mechanism involving the production of dry membranes, cocoon, etc., is perhaps responsible.
It is possible, however, that the eggs of some insects, like those of reptiles, though apparently self-contained, take in water. Thus Wheeler informs us that ants "salivate" over the eggs in their communities and suggests that this saliva may be absorbed. Similarly, Weyrauch reports that the earwig {Forficularia) licks its eggs after laying them; and if this is not done they will not develop.
1 Also in fishes (e.g. Lepidosiren (Carter & Beadle), where it disappears early in development and is perhaps functionless) .
SECT. 6] OF THE EMBRYO 905
And a good deal of evidence exists, indicating that insect eggs, although terrestrial, need a humid environment for proper development. Thus Harukawa obtained the following figures on the Oriental peach-moth :
Relative humidity Percentage of eggs hatching
• 49-4
15
83-1
>5
lOO'O
and it is known that the Trinidad froghopper, Tomaspis saccharina, cannot hatch at all under 90 per cent, humidity. Again, Peterson has shown that Aphid eggs are very dependent on their normal humidity for proper hatching, which is easily stopped by a small decrease in the water-content of the environment {Aphis avenae and Aphis pomi) and Dampf, Hoffman & Varela have shown the same thing for various grasshopper's eggs. (See also Tchang and Andersen.)
It remained for Bodine to show in 1929 that the water-content of the whole system in grasshopper and other orthopteran eggs rose during development. This process was evidently closely associated with the egg's metabolism for increase of temperature accelerated the water-intake. We may therefore have to picture the insects as solving the problem of terrestrial embryonic life, not by providing enough water in the eggs from the maternal body, as the sauropsida do, but by inventing a sort oi deliquescent egg which should absorb atmospheric moisture. In this connection, Peacock has made some suggestive experiments on the eggs of the saw-fly Pristiphora pallipes which normally inserts them in pockets artificially contrived in gooseberryleaves. It seems very probable that water is absorbed by the eggs from the plant, for Peacock found that if the stalk of the gooseberrytwig was immersed in a weak solution of eosin, the dye would pass into the leaves and thence into the eggs. On the other hand, if the eggs were removed from the pockets at the beginning of development they hatched as usual, being able, apparently, to pick up from the air all the moisture they required, and swelhng normally. Swelling, in fact, seems to be a regular occurrence in the development of the eggs of all Tenthredinidae, Cynipidae,and Formicidae. Kerenskihas shown that the eggs of the scarab beetle, Anisoplia austriaca, double their wet weight in a fortnight and that this increase can be at the expense of distilled water, no dissolved substances being taken up, and development proceeding normally. As insect eggs are so small, they no doubt make use of " micro-climates ", for small crevices etc. may have a very different humidity from the main climate in which they exist.
58-2
9o6
GENERAL METABOLISM
[PT. Ill
67. Water-metabolism in Aquatic Eggs
Fig. 238 taken from Kronfeld & Scheminzki's paper illustrates once again the relations we have been discussing in the case of the trout egg. It shows that the maximum intensity of water absorption occurs about half-way through the yolksac free-swimming period. The total water in the larval system rises steadily from fertilisation onwards and also that in the embryo, but there is a distinct loss of water from the yolk. This does not mean that the yolk becomes drier, but is simply a measure of the disappearance of the yolk. Before hatching the loss of water from the yolk is just compensated for by the gain in water of the embryo, showing that at first the system is a closed one. There is here a certain contrast with the frog's egg, for the water content of the embryo plus yolk there begins to rise well before hatching, though Gray asserts that water-absorp- Water in the yolk.
tion begins before hatching in Fig. 238. The vertical lines indicate relative , T i_ 1 intensities of water absorption, and the
the trout too. In the lump- asterisks the maxima of dry and wet weight
sucker {Cyclopterus) Hayes found respectively.
no intake of water until after hatching, but in the Atlantic salmon [Salmo salar) there was a definite rise of about 10 per cent. Kronfeld & Scheminzki fully appreciated the fact that the trout egg contains enough solid but not enough water to make the embryo, and they attempted to show that at the end of the first period a slowing of the growth-rate was perceptible, owing to the inability of the embryo to get sufficient water through the egg-membranes to dilute its solid material. The figures on which they based their growth-rate estimations, however, were rather too few to substantiate this; nevertheless, some statements in Gray's paper appear to coincide with it, and
75
mg 70
]
/
65 60
\("
55
//
50
/ / /
ts
I
/ /
to
35
^EiperiodA/
^1
-Doifensackperiode »
/ *
— Hunger
3D
' \
/
25
- \ \
/ /
20 15 ■10 5
\
1
1 1
, ,9-,
]o W 50
60
tT
80 30 100 110 120 UOTj
Water in the larva. Water in the embryo.
SECT. 6]
OF THE EMBRYO
907
yoLk-sac period -*« hunger—
provisionally it may be accepted. Fig. 239 gives a graph plotted from their data. The percentage growth-rate falls markedly at the end of the first period, only to rise again to a maximum during the freeswimming period. Everything, in fact, points to a resumption of the growth-process as soon as an unlimited supply of water is available, which will only fall off again when the nutriment in the yolk-sac is beginning to be exhausted. Kronfeld & Scheminzki pointed out that the decline in the growth-rate before hatching occurred before all the water in the yolk was finished, as would be expected in view of the osmotic pressure of the latter. They drew attention to ^ Schaper's well-known experi- 5 ment on frog embryos and J larvae, in which he placed them J in salt solutions, and, by thus I holding back the water which | they needed to absorb to form | their tissues, succeeded in in- ^ hibiting their growth. Kronfeld % & Scheminzki confirmed this ^ for the trout in some preliminary experiments. The question of whether growth of the trout fry can continue after the yolk has been used up was left open by Kronfeld & Scheminzki, but Weiss had previously maintained that this could occur, and Podhradski & Kostomarov confirmed Weiss' results on carp alevins at the end of their yolk-sac period. It must be supposed that this growth is either due to the persistence of small amounts of yolk which gradually get used up after the yolk-sac has apparently disappeared, or to the utilisation of muscles or other tissue as nourishment instead of yolk. The latter process is suggested by the work of Krzinecki & Petrov^.
The process of water-absorption by the amphibian larva during its development was studied also by Bialascewicz, who used the unsatisfactory but relatively easy method of measuring volume changes. In all his curves, a temporary reduction of volume is seen about
10 20 30 40 50 60 70 80 90100110120
(0% growth rate wet weight) Embryo KiS<* ° " " "'O' " i
)0 °/» de-growth „ wet .• 1 Yolk l.<j> % r, „ „ dry ., /
Fig. 239.
^ In certain cases, the intake of water by the egg before hatching will mask the loss of dry solids by combustion, if the eggs are weighed in air. Weighing in water, as suggested by Ritter & Bailey, may therefore be a useful method.
9o8
GENERAL METABOLISM
[PT, ITI
Fig. 240.
the second hour; this is due to the formation of the perivitelHne fluid (see p. 784). According to Bialascewicz, this temporary reduction and the formation of the perivitelHne space only occurred in fertilised eggs, as Fig. 240 shows, but perhaps , the difference is quantitative -i rather than quaHtative. In .■ the later stages Bialascewicz measured the volume directly I instead of calculating it from ; the measured diameter, and . his results appear in Fig. 241, taken from his paper, where the average volume of one larva is plotted against the time in days. The shape of this curve is of interest, inasmuch as there is first of all a marked rise, followed by a comparatively stationary period during the last few days of the pre-hatching period, and then by a tremendous rise as soon as the larva has come forth from its jelly, and nothing interposes itself between the tissues and the water but the skin. These data are obviously in close agreement with the findings of Kronfeld & Scheminzki and of Gray. Thus Bialascewicz found that between the 2-cell stage and the blastula stage there was an increase in volume of (average) io-6 per cent., and between the blastula stage and the gastrula stage an increase of 7*6 per cent. Fig. 242 shows the relative speed of volume increase at the different stages expressed in terms of volume increase of 1000 larvae in hourly periods. The higher level at gastrulation
6-5
/
6-0
"e
E
/
5-5
- u
1
5-0
/
4.5
-E
c /
a>
Ic /
c
/
4.0
■^ / X /
3.5
- a)
E 3
y
3.0
"V
"■"--^
2.5
- /
Days 1 1 1 ' 1 1
1 2 3 4 5 6 7 8 9 10 1112 13 Fig. 241.
SECT. 6]
OF THE EMBRYO
909
.E40
was related by Bialascewicz to the presence of the blastopore. The slight diminution in absolute volume between the 50th and 11 8th hours is reflected on the speed curve by a big drop, but after that the curve rises with only minor variations. Both Davenport and Schaper noted an increased growth-rate in the frog larva after hatching, so that the results of all the workers on the frog embryo are in agreement with those of all the workers on the trout. The whole mass of data shows clearly an absorption of water diluting the yolk as it is transformed into the tissues of the embryo. ^20
Bialascewicz also investigated ^ the part played by the jelly of | the frog's egg after hatching as a s source of solid and water for the ^ embryos. Frog larvae are nearly always to be seen hanging on to the jelly with their suckers after having hatched, and Bialasce- ^^' ^'^'^'
wicz rightly thought it not improbable that the jelly might contribute something to the larvae. Measurements certainly supported this; thus the average weights of larvae he found to be as follows :
100 200 300
Hours from fertilisation
Weight in milligrams
8 days 26 days (in distilled water) ... 26 days (in distilled water but plus the jellies)
Wet
9-52 25-24 84-46
Dry
I -08
4-22
This was a striking demonstration of the absorption of water. In the second instance much water had been absorbed, but the dry substance was the same in amount, or rather slightly reduced owing to combustion of yolk, whereas if the jellies were present the dry substance was much increased as well as the water. The loss of dry substance from the larvae was also found by Bialascewicz for the early stages; thus, between the ist and the 4th day, about 5 per cent, of the dry weight was lost. He measured the effect of temperature on the increase in volume of frog's eggs at different stages, and came to the conclusion that temperature had no effect on the amount of water
910
GENERAL METABOLISM
[PT. Ill
Hatching
Fig. 243.
absorbed by the larvae, though the time they took to absorb a definite amount was, of course, made longer or shorter. The effect of rising temperature on the unfertilised eggs was to increase the permeability of their membranes to water, so that, for instance, at 10°, an egg in a definite period would increase its volume by 0-05 c.mm., and at 20° by 0-28 c.mm. But the possibihty that the increase with temperature was really due to the production of an unusually large amount of osmotically active substances in the egg-protoplasm was not excluded. Bialascewicz argued that as the permeability to water is increased five times by a rise of temperature of 10°, and as the development rate is increased only two and a half times, one would expect to find more water in larvae brought up at 20° than at 10°. Since experimentally this was not the case, Bialascewicz concluded that the permeability of the membranes to water was not the determining factor in the absorption of water during amphibian embryonic growth.
Galloway, inspired by Davenport's early work, also made a study of the effect of temperature upon the absorption of water by the developing frog larva. The embryos of Rana sylvestris, Amblystoma punctatum and Bufo americana were subjected to temperatures varying from 6° to 25°, and the water-content estimated from time to time. The results obtained are shown plotted in Fig. 243, whence it can readily be seen that the warmer the environment, the more rapidly did the imbibition of water go on. But Galloway found that at higher temperatures the final amount of water in the body was slightly more than that at lower temperatures although the developmental process up to the point when 75 per cent, water was reached was not so much retarded by lower temperature as the stage representing the maximum percentage of water. (See Table 107.) The effects of temperature, therefore, were more marked on the second phase of the process than on the first. In the first stage, assimilation of yolk and cell-division is prominent, in the second stage, assimilation of water. This is in exact accord with the finding of Bialascewicz, for a period in which imbibition of water is
Time in
days required to attain 75 % water
Highest temp.
2
9
5-5
Medium Lowest
temp. temp.
5-7 20
12 27
7-5 —
SECT. 6] OF THE EMBRYO 911
prominent would thus be expected to be more sensitive to temperature change than one in which it is not prominent. As it is difficuh to see why an apparently physical process should be so much affected by temperature, we must suppose that the absorption of water is regulated by chemical means. Galloway observed a regulatory process at work, in that individuals which were placed at 13° for a week, and then changed to a warmer environment, showed a greater increase of water than those which had been in the warmer environment from the beginning.
Table 107.
Time in days required to attain 95 % water
Highest Medium Lowest temp. temp. temp.
Rana ...... 2 5-7 20 5 28 50
Amblystoma ... g 12 27 21 50 70
Bufo _5^5 7^5 --_ 24 32 ^
Averages ... 5-5 8-4 23-5 13-3 36-6 60
Retardation in \ days, reckoned
from time re- V — 2-9 18 — 23-3 46-6
quired at highest temperature j Retardation simi-"! larly calculated ^ — 53 327 — 175 350
in % ]
The work of McClure provides an interesting commentary on the great absorption of water by the developing amphibian egg. He found that in the early cleavage and medullary groove stages of Bufo and Rana no cell will store trypan blue. But about the time of hatching (9 days from fertilisation) a change takes place and storage of the dye goes on vigorously, as it is taken up from the solution surrounding the eggs. This is probably because the lymph vessels are formed about the 5th or 6th day from fertilisation and can take the dye granules with the incoming water from the periphery to the interior of the organism.
6-8. The Chemical Constitution of the Embryonic Body in Birds and Mammals We may now return to the chick embryo. We have to consider under the heading of general metabolism a variety of data, among which the percentage constitution of the embryo at different stages of its development is among the most important. This leads
912 GENERAL METABOLISM [pt. iii
directly to the question of the absorption curves for the various substances, i.e. the determination of the relative intensities with which various substances are absorbed from the yolk or the nonembryonic parts of the egg. These problems, again, cannot be dealt with without discussing the efficiency with which the raw materials are transformed into the finished embryo at different stages. All these subjects can best be treated in relation to the egg of the hen, for it is there that they have been most carefully worked out; in fact, it is only in the case of the chick, where the embryo can be separated from the yolk from a comparatively early stage, that these problems have been even partially solved.
First, as regards the percentage constitution of the embryo. Table io8 summarises the accurate data which we have on this question. It may be doubted whether in a computation such as this, in which it is desired to compare active strengths of constituents in the embryo, it is advisable to include substances packed away in an immobile form. Glycogen would not come under this category, nor would the adipose tissue of fat depots, but the keratin in the feathers does seem somewhat remote from the balance of reactions in the body. Murray, whose suggestion this was, estimated the amount of feather substance present each day after the 12th, and his figures are represented in Col. 7. He only gave these values, however, as percentages of the body weight, so the actual weights are seen in Col. 8, the corresponding amounts of keratin (assuming the feathers to be 90 per cent, keratin) in Col. 9, and the true protein, excluding the feather protein, in Col. 10. Col. 11 reproduces Col. 6 corrected for the feathers, and finally Col. 12 shows the fat and Col. 3 the carbohydrate in grams per cent, of dry weight.
The graphic representation of this table is to be found in Fig. 244. The curves make up an interesting assemblage, for all three have peaks, and it is important to observe where they come. The carbohydrate one occurs on or before the 5th day, the protein one on the nth day, and the fat one on the 20th day. This is reminiscent of the order in which, as will be seen (p. 993 j, the intensities of combustion run; carbohydrate about the 5th day, protein at 8*5 days, and fat towards the very end of development. The way in which fat overtakes protein at the end of development was first noticed by Murray, and Riddle observed in the pigeons' egg a preferential absorption of fat from the yolk at that time.
Mill
'P '?' " ^ 'P 9 ^v^v^^s^r P ?^ r^ 'P
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914
GENERAL METABOLISM
[PT. Ill
^.000
\\
• Fat ex Protein \ ® Carbohydrate
\ °/
\ y*
-3000
V y^ °
V y"^
y\. °
.r-'V^
-2000
.>K>jW^
tnoo
.1 . 1 1 1 1 1
1 1 1 1 1 1 1 1 1
gms mfims 2 Day3-»5
Fig. 244.
The carbohydrate, protein, fat succession seen in these constitution curves explains Murray's finding that the calorific value of the embryonic tissue increases with age (see p. 947). Before leaving Table 108, some interesting facts which emerge from the values of the last three columns may be briefly mentioned. Col. 13 gives the inorganic material in the embryo in grams per cent, taken from Murray, and together with Cols. 3, 6 and 12, which represent the carbohydrate, protein, and fat, it ought to add up to nearly the whole of the dry weight as experimentally measured. Actually, as Cols. 14 and 15 show, it only adds up to about 90 per cent., leaving 10 per cent, on an average for all the other substances associated with life, lipoids, sterols, cycloses, pigments, waxes, etc. It is interesting to see that this residual value declines steadily during development, as if in the earlier stages there were a higher percentage of substances not included under the heads : carbohydrate, protein, fat, ash. Of course, estimation errors will be included in the residual value, and these will usually lead to slight losses in the substance estimated. When accurate figures become available for the amount of lecithin and cholesterol in the embryo, for instance, it will be interesting to begin the compilation of a balance-sheet of the residuum.
Whether the curves for percentage constitution in the chick embryo will be found to resemble those for other embryos we cannot as yet tell. For the mammalian embryo, however, a certain number of figures are available, though they in no way approach in thoroughness
lCamerer-& Sdldner
Klose
■ Fat 0) Ash -V Protein/ sAsh
o Protein JFehlIng • Fat
DProte;n|Mi a Ash
Months lan Embryo Constitution
Fig. 245.
SECT. 6]
OF THE EMBRYO
915
the data which have been accumulated for the chick. It is possible to construct, from the analyses of FehHng and others on man, Michel on the rabbit, Liesenfeld on the dog and Hayes on the salmon, a table showing the percentage composition of the embryo, and, when
40
p
36
- Q.
32
-70
>,20
•
s;:
■60
8
4
Rabbit
embryo constitution sAsh ]
o Protein fehling ^ • Fat J D Protein]
■ Fat JFriedenthal EiAsh J
Days concepfci
^ 5
Dog Embryo Consbibution ^^^
Liesenfeld, DahmenSiJunkersdorf •
Costantino ♦
Fig. 246.
20 30 40 50 Days conception age
Fig. 247.
the data are plotted on a graph, Figs. 245, 246, 247 and 248 are obtained. Total carbohydrate cannot be considered, for the chick is the only embryo in which this has so far been estimated, and, in any case, it does not form a large part of the total solid there. But it is clear that both in the case of the human and the rabbit embryo there is just that cross-over between protein and fat which we find so markedly in the chick embryo. It is interesting that this was not noticed by the workers themselves, because they did not express their results in percentage dry weight, and all these inter-relations are, of course, obscured, if percentage wet weight is alone taken into consideration, owing to the sharply decreasing water-content. Next it is to be observed that Table 108 demonstrates a distinct lowering of the ash-content in grams per cent, during the development of the chick — this was the discovery of Murray — but the figures plotted in Figs. 245 and 246 do
10 20 30 W 50 60 70 30 40 50 60 70 80 90 100 110 120 -" — Days — ►
Fig. 248.
9i6
GENERAL METABOLISM
[PT. Ill
not show such a fall in the case of the mammalian embryo. Possibly this is due to the fact that we have no analyses of mammalian embryos in the earlier stages, and the earlier, sharper part of the inorganic curve may thus have been missed. Inspection of Col. 13 of Table 108 shows that this decline in ash-content percentage dry weight is in fact more rapid at the beginning than at the end of development. Some fragmentary data for the Jersey cow embryo contained in the paper of Moulton, Trowbridge & Haigh, do not seem to show any change between the 1 85th day of gestation and birth :
Percentage
Days of gestation
Fat
Protein
Ash
185 232
Birth
14-6 14-0
69-0 69-0 69-0
Il-O
17-4 17-4 ib'O
'arying balance of the chemical
Popov has also made analyses of cow embryos, but I have not been able to gain access to his data.
Another way of looking at the constituents of the developing embryo is to enquire whether the ratios between any two of them change with age. This method was first used by Murray. "Before undertaking my experiments", he said, "I was impressed by what seemed to be a natural scale or gradient, as judged by various criteria, of the chief groups of substances under consideration, namely, salt, carbohydrate, protein, and fat. A tentative prediction was considered, that the following ratios would be found to decrease with age during ontogeny water/ solid, inorganic/ organic, carbohydrate/protein and protein/fat." The analyses which Murray and Needham subsequently made confirmed this prediction in every particular. Murray himself attempted to get the carbohydrate/protein ratio by estimating the amount of glycogen in the chick embryo at different stages, but
400
300
..j^ Carbo hydra be
Protein + Probe! n • Fat
Days-*5
10 Fig. 249.
SECT. 6] OF THE EMBRYO 9^7
this was an unfortunate substance to choose as a representative of the carbohydrates, in view of the phenomenon of the transitory Hver^. When I calculated this ratio on the basis of analyses of total carbohydrate, a definite decrease appeared, just as was expected. The bundle of curves which the plots of the ratios against the age give is shown in Fig. 249, and it is seen that they all pass downwards together.
Some of the minor points in this graph are worth considering. The decHne in the ash/organic substance ratio, for instance, reminds one of the similar dechne in the case of the frog embryo (see Fig. 230), and of Spek's suggestion that the velocity of cleavage of cells may depend, at any rate partially, on the concentration of electrolytes in which they find themselves. Perhaps we catch a glimpse here of a mechanism which controls cleavage velocity, for if by some means it were arranged that the ash-content of an embryo should fall with age, then the other factor might fall likewise, and the growth-rate would follow suit.
6-9. Absorption-mechanisms and Absorption-intensity
So far we have been considering the constitution of the embryo and how it changes with age, but the next question concerns the relative intensity of absorption at different times during its development. Up to the present time it has only been possible to make a start in this direction with the avian embryo, although morphologically a good deal is known about the various methods of absorption of the materials stored in the egg, and before considering the absorption intensity of the chick, something may be said about the absorption processes of other embryos. The principal treatment of this subject is that of Peter. It is evident that a fundamental distinction will here arise between holoblastic and meroblastic eggs, for if cleavage is complete and the whole egg divides into equal or nearly equal parts from the very beginning, the yolk will also get divided more or less equally, and being there already will not require any special means of transport into the constituent parts of the embryo. On the other hand, where cleavage is partial, and the vegetal pole of the egg does not divide at all, or where, as in the case of birds, 90 per cent, of the egg is vegetal pole, then mechanisms of various degrees of com ^ See Section 8-5.
9i8
GENERAL METABOLISM
[PT. Ill
plication have to come into play to supply the cells of the embryo with their yolk.
It is probable that the amount of yolk furnished to the egg by the parent has a good deal to do with determining whether its cleavage shall be complete or partial. Thus Peter's table (Table 109)
Table 109.
Diameter of
Equal cleavage
egg in mm.
Mouse (Mus) ...
o-o6
Lanceolet (Amphioxus)
01 - 0-13
Man (Homo)
0-15- 0-20
Rabbit {Lepus)
0-l8- 0-20
Unequal cleavage
Toad (Bufo)
0-6 - 0-15
Lamprey {Petromyzon)
I-I - 1-2
Newt {Triton)
1-6 - 0-2
Frog {Rana)
2-0
American bowfin (Amia)
2-5 - 3-0
Sturgeon (Acipenser)
2-8
Australian lungfish (Ceratodtis)
2-7 - 3-0
Obstetric toad {Alytes)
3-0 - 5-0
African lungfish (Protopterus)
3-5 - 4-0
Salamander {Salamandra)
3-5 - 5-0 6-5 - 7-0
South-American lungfish (Lepidosiren)
Caecilian (Hypogeophis)
7-0 - 8-0
Caecilian [ichthyophis)
7-0 - 8-0
Partial cleavage
Sea-perch (Senanus) ...
0-8
Herring (Clupea)
0-9 - i-o
Perch {Perca)
1-4
Duck-billed platypus (Ornithorhyncus)
2-6
Anteater {Echidna)
3-0 - 4-0
Garfish {Lepidosteus)
3-0 - 5-0
Trout (Trutta)
4-0 - 5-0
Trout {Salmo)
6-0
Lizard (Lacerta)
9-0
Snake {Trochilus)
13-3
Dogfish (Pristiurus)
15-17
Hagfish {Bdellostoma)
20-30
Torpedo {Torpedo)
20-25
Adder {Pelias)
21-25
Hen {Callus)
35
Alligator {Alligator)
40
Ostrich {Struthio)
105
Porbeagle shark {Lamma)
220
shows that the larger the tgg the more likely it is to cleave only partially and to require special absorption methods, and the same thing is shown (roughly) by the relation between the size of the egg and the ratio between macromere and micromere sizes. In other words, the more yolk the egg has, the larger the yolk-laden macromeres will be compared to the micromeres. ±
SECT. 6]
OF THE EMBRYO
919
Size of egg
Ratio of micromeres to macromeres: ilx
(diam. in mm.
X
o- 1-0-3
I-I-I-2
8i
I-6-2-0
1-33
1-4
11
3-0 3-5-5-0 6-5-7-0
2-5 4-5 3-0
Amphioxus
Petromyzon
Triton ...
Rana
Amia
Adpenser
Ceratodus
Salamandra
Lepidosiren
The nature of the yolk is also believed to influence to a large extent the form of cleavage. Thus yolk with large formed elements probably necessitates unequal divisions, and Hertwig was able by centrifuging, to make the frog's egg, which normally divides totally but unequally, divide partially.
Table no.
Absorption
Direct
By the
through the
intestine
division of
By the
and the
the blasto
Mero
Yolk
yolk-sac
pancreatic
meres
cytes
cells
epithelium
juice
Urodele amphibia
+
_
_
_
(e.g. Salamandra)
Gymnophiona (e.a
^ +
Unequal
Ichthyophis)
cleavage
Protospondyli
+
+
~
(e.g. Amia)
Aetheospondyli
+
+
_
- "
(e.g. Lepidosteus)
Cyclostomes (e.g.
+
+
—
—
—
Bdellostoma)
Selachians (e.g.
+
+
—
—
+
Scyllium) Telosteans (e.g. Salmo)
+
Partial cleavage
Saurians (e.g.
+
+
+
+
Lacerta)
Aves (e.g. Gallus*)
+
+
+
Monotremes (e.g.
+
+
Echidna)
J
A complete account of the histology
of yolk-absorption in
the chick v
vill be found
in the monograph
of Remotti.
The yolk
absorption
in cephalopods is very
peculiar (see
Portm.ann & Bidder).
The special absorption methods necessitated by partial cleavage are shown in Table no. In the simplest type, shown by Salamandra, the macromeres gradually hand over their contents to the rest of the embryo. The appearance of merocytes and yolk-cells, situated
920 GENERAL METABOLISM [pt. iii
in, and absorbing, the yolk, complicates the process further. Then, as the mass of yolk becomes gradually more and more enormous relative to the embryo, the walls of a special yolk-sac take on absorptive functions, and in birds, for example, become of great importance. The monotremes show the abandonment of the intermediate methods^.
We may now return to the rate of absorption of the yolk in the hen's egg.
An absorption-coefficient could not be calculated from the constitution figures only, for the rate of combustion of certain substances and the rate of transformation of others might, and in fact actually does, vary considerably. The only way to find out the relative absorption intensity of the various substances of the food-supply by the embryo is to make a large number of analyses and special calculations. So far the chick is the only embryo for which this has been possible. Murray's work provided the greater part of a sound foundation for these computations, and I made them in 1926 and 1927.
We may take first the absorption intensity of protein throughout development. To calculate it, it was necessary first of all to know the amount of true protein inside and outside the embryo on each day of incubation, and this was obtained by making various corrections ; thus from the total nitrogen of the embryo were subtracted {a) the lipoid nitrogen, calculated from the work of Plimmer & Scott, and of Masai & Fukutomi, (b) the purine nitrogen, calculated from the work of LeBreton & Schaeffer, and {c) the water-soluble nonprotein nitrogen, taken from the work of Needham. For the total protein nitrogen outside the embryo, the figures of Sakuragi were used, after being corrected for the use of trichloracetic acid instead of acetic acid and heat. The way these data were used is shown in Table iii. Columns 2, 3, and 4 concern the embryo and need no special remark.
^ A most interesting sidelight on the nature of yolk-absorption is given by the work of Newman on hybrids of the minnows Fundulus majalis and Fundulus heteroclitus. The former fish lays eggs 2-7 mm. in diameter, the latter 2 mm. An F. majalis ? x F. heteroclitus ^ cross gives after about a month a hybrid embryo resembling the paternal species, and too small for its yolk, i.e. unable to assimilate it. Although in the presence of excess food, its absorption-mechanisms seem incapable of dealing with it and the hybrid, sinking to the bottom, dies before it can hatch. The opposite hybrid (where the egg is small) hatches small, unable to reach the large size of its paternal origin, but is quite viable (cf. the genetic differences in avian yolk-absorption-rate mentioned on p. 940).
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59-2
922 GENERAL METABOLISM [pt. iii
Cols. 5 to 9 deal with the egg as a whole. Col. 5 gives the apparent protein nitrogen in the whole egg for the intermediate periods; it is obtained by considering the period "5th-to-6th-day", for instance, as if it were 5-5 days, and taking the average of the values for the 5th and 6th days. Col. 6 performs an exactly similar service for the true protein nitrogen in the embryo. It will be clear that, since Col. 5 is for the whole egg, the subtraction of Col. 6 from it will give figures for the remainder, the non-embryonic part of the egg. The result of doing this is shown in Col. 7, which represents the protein nitrogen in the remainder of the egg for the intermediate periods, corrected for the lipoid nitrogen outside the embryo, but still including the false protein nitrogen inside the embryo. Cols. 8 and 9 perform this final adjustment. Col. 8 shows the lipoid and purine nitrogen inside the embryo, calculated for the intermediate periods, and Col. 9 the true protein nitrogen in the remainder of the egg at any given moment during development.
Cols. 4 and 9 are now the ones on which attention must be focused. If Col. 4 is expressed as a percentage of Col. 9, we shall be finding what 100 mgm. of true protein nitrogen outside the embryo hand over during each interdiurnal period to the embryo. We shall have the milligrams absorbed each day in percentage of what each day remains to be absorbed.
It is clear from a summary inspection of the figures in Col. 10 that this value is not very illuminating. It only shows the gradual increase in size of the embryo. But if now this value is expressed as percentage of wet and dry weight, we shall be calculating what 100 mgm. of protein nitrogen hand over to 100 grams of embryo throughout development, and we shall be able to observe the varying intensities of the progress. Cols. 11 and 12 simply give the weight data of Murray and Needham calculated for the interdiurnal periods. Cols. 13 and 14 give the final results.
They are shown graphically in Fig. 250. It is seen that the absorption of protein, in the first few days of development very rapid, falls off exceedingly between the 6th and loth days, to rise again, however, to a high peak on the 15th or i6th day. After that point it again falls to about its previous level. The most interesting thing to notice is that, as far as protein is concerned, there is no correlation whatever between absorption and combustion. The peak of protein combustion (see p. 993), which occurs between the 8th and the 9th
SECT. 6]
OF THE EMBRYO
923
%
days, comes just in the trough of protein absorption. The two processes seem to be entirely distinct, as is clearly shown by the broken line representing protein combustion in Fig. 250. The 15th day peak corresponds remarkably well with the fact that about that time the vascular bag or "avian placenta" of Duval is formed by the fusing ends of the allantois. The accelerated absorption of protein from the egg-white shows itself as clearly in Fig. 250 as does the accelerated diminution of bulk of egg-white in Fig. 199. It is also interesting to note the effect of relating absorption to dry and to wet weight. The increasing dryness of the embryo has the result of minimising both the trough at the 7th day and the peak at the 15th.
The process by which a knowledge of the absorption curve for fat was derived differed in no way from what has already been seen in the case of protein, except that it was less complex, fewer corrections to the basic values being necessary. Unfortunately, the weight resulting curves cannot claim the same degree of accuracy as may be accorded to those for protein, for an at present unresolved discrepancy exists in the literature between the measurements of egg-fat. As will be seen in Section 8-4, from the 7th to the 14th day the fat lost, as determined by the averaged chemical analyses, is considerably in excess of that lost as determined from the carbon dioxide output, even assuming that all the carbon dioxide was derived from fat, which is not true. But it is probable that this error, which is certainly real and of some theoretical importance, does not upset the general shape of absorption curves calculated disregarding it. When its nature is cleared up, the fat absorption curve may have to be revised.
In Table 112, the various stages of the calculation are set out. Columns i to 4 need no comment. In Cols. 5, 6 and 7 are shown, first, the fat in milhgrams per whole egg, calculated for the interdiurnal periods from the experimental data of Eaves; Idzumi; Murray; and Sakuragi ; secondly, the fat in the embryo calculated for the same
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Absorption curves
Fig. 250.
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SECT. 6]
GENERAL METABOLISM
925
lapses of time, and, finally, the difference between the two, in other words, the milligrams of fat present in the non-embryonic part of the egg in the intermediate periods. In Col. 8 is found the miUigrams absorbed each day expressed in percentage of what at that day remains to be absorbed. This is the column representing the amount of fat handed over to the embryo out of 100 mgm. of external fat between each two days. Cols. 9 and 10 express the same value only related to 100 gm. of embryo, wet and dry. When Cols. 9 and 10 are plotted upon a graph, very interesting curves are seen. Fig. 251 shows that the absorption curve for fat rises and falls in much the same way as that for protein. It possesses a peak about the loth day, and another rapid rise about the i8th^. What is at once noticeable is that these periods do not synchronise with the period of pre-eminent fat combustion. The 15th day is the centre of the period at which the respiratory quotient is 0-73 (Bohr & Hasselbalch), yet at that very day there is a distinct trough in the curve of fat absorption. We see, then, that, as with protein, so with fat, there is no chronological relation between combustion and absorption.
Secondly, it may be pointed out that the observation of Gage & Gage, that Sudan III eggs do n'ot give coloured embryos till the middle of development, fits in well with the absorption curve now found. Up to the 8th day absorption of fat goes on very slowly.
The third point of interest is this. Although the curve for fat rises and falls in much the same way as that for protein, it does it at quite different times ; it does not resonate with it ; protein peaks correspond to fat troughs and vice versa. This is well seen in Fig. 252, which shows the averages between wet and dry weights in each case. It should
^ The only other absorption-curve which has been studied is that for lead (Bishop). It shows a marked trough at the 15th day, corresponding with Fig. 251. This is interesting because Bishop concluded on quite other grounds that lead is present in yolk almost wholly in combination with lecithin (e.g. 99 % of it is ether-soluble) . Lead lecithin and lead oleate can, indeed, be isolated from yolk.
© 00
5 10 15
Dry Wet
Absorption curve for fat
weight
Fig. 251.
926
GENERAL METABOLISM
[PT. Ill
be well understood that Fig. 252, as far as absolute values go, is meaningless: it would represent the absorption of embryos 50 per cent, less rich in water than they really are: but it demonstrates more clearly the relationship of fat and protein. In the last stages of development, "it is as if the protein were displaced by fat", says Murray, speaking of fat storage, and this is just what Riddle found in his studies of the yolk in pigeons' eggs. At the end of development there was a marked preferential absorption of fat. Thus from the I St to the 6th day very Httle fat is being absorbed, but a great deal of protein, from the 6th to the 12th day exactly the reverse holds, from the 12th to the 17th day the protein again takes the prominence, while after that time the fat once more overcomes the protein. The rhythms of the two absorption curves are entirely distinct.
At first sight, it would not nificance of this. One way of expressing it would be to say that the cells of the blastodermal blood-vessel walls which collect the nourishment from the yolk and the white have periods at the ends and beginnings of which the properties of their membranes alter. At one time they will admit fat-soluble substances in predominance, at another time water-soluble substances. Putin this way, the phenomenon im
seem easy to estimate the sig
Oays •— > 5
Fig. 252. The reason for the ungraduated ordinate is explained in the text.
mediately reminds us of the state "of affairs seen by many observers in the egg of the sea-urchin where there are periods of permeability to water-soluble substances, and other periods of permeability to fat-soluble substances. At one point the resistance to alcoholchloroform-ether cytolysis is remarkably high, at another it is extremely low. A similar curve can be prepared for a water-soluble substance, such as potassium cyanide, and its summits are seen to correspond to the troughs of the previous one. That rhythms of permeability to fat-soluble and water-soluble substances should be present in the single developing egg-cell of the lower animals, and should then appear again in the complicated avian organism during its ontogenesis is certainly possible, and, if real, of considerable
SECT. 6] OF THE EMBRYO 927
interest. It must be stated, however, that the rhythms of permeabiUty and susceptibility during the cleavage of echinoderm eggs are not regarded as significant by some investigators, who ascribe them to purely mechanical causes associated with the changing shape of the embryo during the cleavage stages. An account of them will be found in the Section on resistance and susceptibility.
How does the absorption intensity of carbohydrate vary during the development of the chick? The study of the carbohydrate metabolism of the hen's egg which I made in 1927 provided the data which were requisite for the answering of this question. Exactly the same procedure which had been previously applied to protein and fat was applied to carbohydrate; the calculations appear in Table 113, and the resulting curve in Fig. 252 alongside the curves for protein and for fat. The difficulty here was to assess the amount of glucose combusted during each period. This can evidently be no more than a rough approximation, for no accurate data exist on carbohydrate combustion, nor is it easy to see how they could be obtained, since sugar is burned completely away to carbon dioxide and water, leaving no end products whose concentration can be measured. Fiske & Boyden pointed out that the combustion of 100 mgm. of glucose would produce 75 c.c. of carbon dioxide, whereas in the first five days the embryo only produces 10 c.c. and Col. 3 was constructed bearing this in mind. All the carbohydrate lost cannot have been combusted. Col. 4 gives the sum of Cols. 2 and 3, i.e. the amount absorbed in each period, and Col. 5 shows the amount remaining outside the embryo. In Col. 6, Col. 4 is expressed in percentage of Col. 5; in other words, this gives the amount absorbed each day in percentage of the amount remaining to be absorbed. Cols. 7 and 8 give the weights of the embryo, dry and wet, calculated irom Murray's data and some of mine. Cols. 9 and 10 show the intensity of absorption of carbohydrate calculated for wet and dry weight.
The two questions which this curve answered were {a) whether there was any relation of simultaneity between the absorption and combustion of carbohydrate, and {b) whether there was any likeness between the absorption curves for protein and carbohydrate, for, if so, the conception of rhythmic permeability changes on the part of the cells of the blastodermal blood-vessels would receive support.
The period of predominance of carbohydrate combustion is belie\"ed to be in the first week of development, and from Fig. 252 it
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SECT. 6] GENERAL METABOLISM 929
may be seen that the absorption intensity is then at its highest. In this way carbohydrate differs from protein and fat.
During the first 6 days the absorption of carbohydrate and protein (both "water-soluble substances") is very high, while that of fat is very low. From the 6th to the 13th days the intensity of absorption of fat is high, and protein and carbohydrate are low. From the 13th to the 17th days the absorption of fat again drops, and that of protein rises, but carbohydrate does not accompany it; on the contrary, it remains very low, though moving slowly in an upward direction. At the i8th day there is another sharp cross-over between protein and fat which is not shared by carbohydrate.
The behaviour of carbohydrate gives, therefore, some support to the theory that changing types of permeability in the yolk-sac cells are responsible for these effects. In the earlier periods when the absolute amounts of carbohydrate being absorbed are comparable with the absolute amounts of fat and protein being absorbed, the protein-carbohydrate curve does tend to be the reciprocal of the fat curve, but later on the carbohydrate drops out of the relationship, and pursues an uneventful course of its own. Thus on the 6th day the embryo absorbs 5 mgm. of carbohydrate, 6 of protein and 2 of fat, while on the 1 6th day it absorbs 1 1 of carbohydrate, 449 of protein and 333 of fat. During the period, then, when the absorption of carbohydrate is at all equivalent to that of the other food-stuffs the relations predicted by the hypothesis hold in practice.
It will be worth while to compare carefully the curves for absorption intensity in the chick embryo, with those for its constitution, for the correlation between constitution and absorption is quite close. When the intensity of absorption of carbohydrate is at its highest, then there is more carbohydrate in the embryo than at any other time. When the intensity of absorption of protein is passing its second peak the constitution curve has also its peak. When the intensity of absorption of fat has risen to its highest level the amount of fat in the embryo has done so too.
On the other hand, there are two points where the correlation is not obvious. The first peak on the fat absorption curve has no corresponding inflection on its constitution curve, and the initially high absorption intensity of protein has nothing to correspond with it. There is, of course, no a priori reason to expect close agreement between the peaks on the absorption and constitution curves.
930 GENERAL METABOLISM [pt. iii
When one shows that a certain amount of one kind of building-stone has entered the embryo and the other shows that it is not to be found there in the form in which it went in, the inference may be drawn that it has been changed into some other material of architectural or energetical value. Now not all the fat entering the embryo between the 8th and 1 1 th days appears there immediately afterwards as fat. One cannot help being reminded of the coincident transformation of fat into carbohydrate, which will be discussed below in Section 8-4. At that time some 40 mgm. of carbohydrate rather suddenly appear from somewhere, and at the same time there is a quantity of missing fat of about the same order. That these transformations appear to take place in the extra-embryonic part of the egg is no objection to the view that they really take place in the embryo, for the circulation in the egg is fairly efficient at that time, and could readily bring the reactants in this change to and from the embryo. In fact, just as we are accustomed to assume that absorption must precede combustion, so it would be logical to assume that absorption must precede fat-carbohydrate transference. There would then be no reason why the products of this reaction should remain in the embryo. Although the absolute amount of substance thus transformed is small enough yet relatively to the size and constitution of the embryo at the time when it takes place, it would account for the peak in the absorption curve. If these arguments are sound, then we must picture an increased passage of fat into the embryo towards the midpoint of development, having for its goal the formation of some new carbohydrate. This will then return to the yolk, and in a short time give rise partly to glycogen in the transitory liver and partly to some other form of combined sugar.
It will have been noticed that, though the peak in the protein constitution curve corrected for feather protein shows some correspondence with the protein combustion curve (11 days : 8-5 days), it would be rather more fitting when comparing the constitution curve with the absorption curve to use the uncorrected values. When this is done, there is revealed an exact correspondence ( 1 6 days : 1 5 days).
The method of calculation which has proved so successful in comparing relative absorption intensities in the case of the chick embryo unfortunately cannot be applied to mammals, for we do not know, and at present have no means of knowing, what the active mass of
SECT. 6] OF THE EMBRYO 931
the foodstuff provided by the maternal organism amounts to. We cannot, therefore, enquire how many grams 100 gm. of raw material hands over to 100 gm. of formed embryo in a given time. Consequently the data we have on the absorption of material by the mammalian embryo are fragmentary, and are almost entirely concerned with the problem of how much the mother on a given diet can afford to store away in the developing embryo.
Thus Magnus-Levy calculated that the average daily deposition in the human foetus for the last hundred days of its development represents not more than 3-0 gm. of protein, 3-5 gm. of fat, and about 0-7 gm. of ash. His table was as follows:
Weight in gm.
t ^ ^
Nitrogen
Fat
Ash
Wet Dry
(gm-)
(gm.)
(gm.)
7 months embryo
950 155
16
26
26
Embryo at term
3200 925
62-5
350
92-5
Increase in loo days ...
2250 770
46-5
324
66-5^
Increase per day
22-5 7-7
o-4b*
3-24
0-665
I.e. of protein 3-0.
It is not possible to enter here into the question of whether the absorption of raw materials by the mammalian embryo entails loss from the maternal tissues or not. The question is a very complex one, and reference should be made to the reviews of Magnus-Levy; Feldman; Hoffstrom; Eckles; Harding; and MurUn, and to an anonymous one which, although now some twenty years old, is worth consulting. Mention maybe made, however, of the interesting case studied by Rubner & Langstein, where a 7-months embryo was born, and was available for the study of the absorption and retention of the food. The infant weighed 2050 gm. at birth, but 8 days afterwards its weight had diminished to 1900 gm., though after that it began to increase by about 28 gm. a day. When the experiments began it weighed 2360 gm. During the next 11 days it retained 50 per cent, of the nitrogen in its milk, though at this time, which would have corresponded to the 8th month of pregnancy, the addition of protein to the child amounted to only a half of that computed by Hoffstrom for the foetus of the same age. The diet contained 126 Cal. per kilo body-weight, of which 73 were used for heat production (973 Cal. per sq. metre per day) and 53 were deposited in the infant. Altogether 42 per cent, of the Calories taken in with the food were retained for growth.
932
GENERAL METABOLISM
[PT. Ill
It is interesting to enquire what is the absorption rate of the embryo at the different stages of its development In the case of the mammal this is again impossible, for we know nothing quantitative about the substances burned by it. Its storage rate can, of course, easily be calculated from the figures of Michel or Fehling, thus :
Months
5
9
Weekly percentage increment of nitrogen storage in the human embryo
0-22
0-20
O From dry wt.and oxygen
consumption ® From chemical analyses
•7- 0) •6I--0
but such a calculation tells us nothing more than that the percentage growth-rate is decHning, a fact already well known. The true absorption rate has only been calculated for the chick embryo. Murray obtained it in 1926 from his determinations of carbon dioxide excreted and oxygen taken in by the chick embryo from the 5th day of incubation onwards by simply adding the rate of storage in terms of weight (see p. 384) to the rate of respiratory exchange, likewise in terms of weight measured by oxygen usage. His values are shown in Fig. 253, where the rate of dry solid absorption per gram of embryo (dry) per day is plotted against the age. Evidently the curve falls rapidly the equation:
Solid stored + solid burnt = solid absorbed
can also be demonstrated in another way, namely by adding together the results of all the chemical analyses of protein, fat and carbohydrate and ash. Of these we have very reliable figures for the amounts of carbohydrate, protein and fat stored, and for the amount of protein combusted, but the amounts of fat and carbohydrate combusted have never been directly measured, and must therefore be approximated. Nevertheless it was of interest to calculate the theoretical absorption
E - cr>
Days -^5
10 Fig- 253.
But the use of
SECT. 6] OF THE EMBRYO 933
curve from these biochemical data, and see how well they agreed with the curve obtained by Murray. The curve so resulting is placed beside Murray's in Fig. 253. His smoothed curve is S-shaped, but that calculated from the chemical analyses tends rather to be uninflected. Considering the many operations involved in the establishing of the chemical curve the agreement is good, but it is not possible to decide which curve is the more reliable.
The consumption of food, as Murray pointed out, is enormous in the early periods. On the 6th day, for instance, the embryo absorbs over its own mass of dry solid, which would be equivalent to an adult man eating about 1 50 pounds of food per day. During the time between the 6th and the i8th days of incubation, this rate falls to about a quarter of its original value. According to Lotka, a mature meadow-lark consumes about 6-6 of its own weight in one day, so that the fall in absorption rate must continue for a considerable time after hatching.
A few words may be included at this point about the routes of absorption of material by the embryo, for the subject is not wholly of morphological importance. Wislocki injected trypan blue into the air-chamber, yolk-sac, allantoic and amniotic sacs, and allantoic mesoblast at 11 days' incubation. On opening 2 days later, no staining of embryo or membranes was observed after injections into the air-chamber, but the trypan blue in the yolk was absorbed vigorously by the epithelial cells lining the interior of the yolk-sac. Eventually the dye penetrated the basement membrane on which the endoderm rests, and reached its final destination in groups of cells surrounding the rich venous network in the yolk-sac wall. It did not pass into the vessels and so to the embryo, but it well illustrated how other substances would do so. In spite of the connection between the yolk-sac and the intestinal lumen through the vitelline duct no trypan blue passed into the embryo by that means. From the allantoic cavity no absorption of trypan blue occurred. From the amniotic cavity there was some passage of the dye into the intestines but no other absorption, Hammar next reported a few generally similar results with neutral red & cresyl blue, and Hanan later continued these studies with trypan blue and methylene blue. Making injections into the air-space and examining the egg at varying periods subsequently, he found that as a rule the stain appeared in the eggwhite, the allantoic Hquid and membrane, and in the embryo,
934 GENERAL METABOLISM [pt. m
especially the mesonephros — but not in the yolk, the amniotic liquid, the amniotic membrane or the lumen of the intestine, although if the egg was not opened for a week or so after injection, the dye would appear in the second group of structures also. The fact that trypan blue, although present in the egg-white at a time when a marked absorption of water is going on by the yolk, does not enter the yolk, throws a light on the properties of the vitelline vessels. Similar work was done by Latta & Busby^. Zaretzki also noted that methylene blue does not enter the yolk from the albumen. After the opening of the sero-amniotic duct about the 12th or 13th day of incubation, the amniotic liquid includes flakes of the protein from the albumen-sac, stained blue, and these enter the chick's intestine. Wislocki's failure to obtain entry of the dye into the egg from the air-space must have been due to the use of too small injections, for Hanan found that dye appears in the mesonephros about i^ hours after injection into the air-chamber.
6-10. Storage and Combustion : the Plastic Efficiency Coefficient
Absorption of raw materials for embryonic growth can be considered in several ways. Related to the embryonic substance already formed, it gives absorption rate; related to the embryonic substance already formed and to the raw material remaining, it gives absorption-intensity; related to the amount of substance being simultaneously combusted, it gives storage efficiency. We may now consider the last-named of these entities. The substrates of the absorption process divide perforce into two parts, one of which is stored and the other combusted as a source of energy.
The degree of efficiency with which the transference of yolk and albumen into flesh and blood is effected may most conveniently be expressed by an efficiency coefficient. This corresponds to the " Rendement materiel " of Henri, the "Coefficient d'utihsation" of Terroine & Wurmser, the " Coefficient economique " ofPfeffer, and the "Plastic equivalent" of Waterman. The best name for it would seem to be "Plastic efficiency coefficient" (P.E.C. for short), for this shows that it has nothing to do with energy content or expenditure, and explains that it is a measure of efficiency of transfer of matter. It may be described as the ratio:
Dry weight of embryo/Dry weight of absorbed solid,
1 Fazzari, too, has studied the absorption of iodine after injection into the yolk.
SECT. 6]
OF THE EMBRYO
935
Plastic efficiency coefficient O Gray : Cumulative © Needham : Incremental
and naturally shows the relative cost in grams of soHd of building the embryo. The higher the efficiency coefficient, the smaller the amount of burnt substance in relation to stored substance.
Gray, in his memoir on the chemical embryology of the trout, already referred to, found that its average plastic efficiency coefficient (P.E.C.) was 0-63. He worked it out for the chick from Murray's data in a cumulative way, but a more instantaneous picture is given when it is calculated on a daily basis, as I showed in 1927. How expensive is it on each day of development to build what is built on that day? It is evident from Fig. 254 that both curves fall and then rise, and the lag in the cumulative one is not significant, for p.^.c. each day's point bears, as it were, in itself the effects of the previous days. The incremental P.E.C. shows the instantaneous change.
There must be some significance in the deep trough through which the curve passes between the 7th and 1 2th days. Evidently at that period development is most expensive; the amount of burnt substance is greater relatively to the amount of stored substance then than at any other time. This suggests a correlation combustion, which is indeed very as may be seen from the vertical line that we have here to deal with action is difficult to resist responsible.
The average P.E.C. for the whole of the chick's development is 0-68. It is interesting to enquire which of the food-stuffs contribute principally to this degree of efficiency. Knowing that fat is the chief food-stuff combusted, and that protein is the chief architectural material, it would be natural to predict that the most efficiently stored substance would be protein. The exact figures follow.
Days-* 5
Fig. 254.
with the intensity of protein exact — in fact, strikingly so, in Fig. 254. The inference an effect of specific dynamic but probably more than one factor is
936 GENERAL METABOLISM
Table 114.
Mgm.
P.E.C.
% of total food-stuff combusted
Carbohydrate stored burnt
1071 25J
0-82
5-6
Protein stored burnt
2986) 69I
0-98*
3-02
Fat stored
burnt
Total solid stored
1 7001 2110,1
4793
0-43 Av. 0-68
91-4
Dry weight of embryo at 19-5 days approx.
5000
Fiske & Boyden by independent reasoning, arrived at 0-96 for this value, and Sznerovna's data give 0-92.
Out of 100 gm. of protein in its diet, then, the embryo can store away 98, out of 100 gm. of carbohydrate 82, but out of 100 gm. of fat only 43. In the case of animals such as the trout, which burn large amounts of protein, the "foodstuff P.E.C." will be very different.
The average P.E.C. can be calculated for a number of other organisms:
Organism
P.E.C.
Investigators
yLould {Aspergillus niger) Silkworm {Bombyx mori) embryo Trout {Salmo fario) embryo Frog {Rana temporaria) embryo
059 0-59 0-63 0-58
Terroine & Wurmser
Farkas
Gray
Faure-Freiniet & Dragoiu
The average efficiency of transfer of the material yolk and white into the material of the embryo seems, then, to be constant for a wide variety of species. But it is now generally recognised that these average figures give very little information about what is actually going on, and it is therefore necessary to enquire how the P.E.C. varies during the developmental process. We ha\e no data for this in the case of any other animal than the chick, except Gray's work on the trout. The growth-rate of this embryo falls off during the later stages of its development, and as its metabolic rate (respiratory intensity, or maintenance intensity) was found by Gray to remain constant, then evidently its efficiency must get smaller as it grows. This relative constancy in the metabolic rate differentiates it sharply, of course, from the chick (see Figs. 126 and 143). Gray accordingly computed the P.E.C. for each successive half-gram of yolk, with the following results:
0-76
0-54
0-71
0-43
0-65
o-ig
SECT. 6] OF THE EMBRYO 937
Hayes, on the other hand, found exactly the opposite on the Atlantic salmon; a rising instead of a falling P.E.G.; 0-36 before the looth day and 0-52 afterwards. Obviously a further extension of our knowledge of plastic efficiency coefficients would be very desirable.
The effect of temperature upon the P. E.G. raises an interesting problem. For the mould, Aspergillus niger, Terroine & Wurmser in their classical paper, could find no difference, thus
Temperature ° C. P.E.C. 22 0-44
29 0-43
36 0-43
38 0-44
They therefore concluded that although in a given time the amount of mycelium formed would be less at the lower temperature, the amount of material combusted would be correspondingly less. Combustion would always go parallel with storage. Rubner found much the same thing in the case of the growth of Proteus vulgaris, and Terroine & Wurmser were led to formulate the general rule "that the quota of material and energy utilised during a given piece of biological work, does not vary appreciably within the limits of temperature compatible with life, although the rate at which the work is done will, of course, vary greatly with temperature". In general this conclusion was supported by the experiments of Terroine, Bonnet & Joessel with germinating seeds (for further discussion of this work see Appendix iv)
r^" — .- - r
Temperature ° C.
P.E.C.
orghum
33
0-54
17
0-77
,entil ...
3?
0-56
18 II
0-57 0-56
jnseed
...
21
0-89
II
0-83
irachis
30
0-85
17
0-86
Next Barthelemy & Bonnet examined the development of frog embryos at various temperatures, and obtained for the P.E.C. the following results :
^ Temperature ° C. P.E.C.
1st series ... 8 0-88
10 0-51
14 0-54
21 0-49
2nd series ... 8 0-85
10 0-79
14 0-84
21 0-83
938 GENERAL METABOLISM [pt. hi
Clearly the second of these series was in agreement with the law of Terroine & Wurmser, but the first was not. In the first series the higher the temperature the lower the efficiency, i.e. the more the relative amount of combustions and the less the relative amount of storage (although the figures do not show a regular progression). Parallel experiments were made by Gray on the trout embryo, with the following results :
Temperature ° C.
P.E.C
1st series
lO
0-56
15
0-50
2ncl series
5
0-49
9
0-43
17-5
0-34
Gray emphasised these figures, which in his opinion demonstrated that the combustion processes had a higher temperature coefficient than the storage ones, i.e. that development was most efficient at low temperatures, for then storage was carried on to the accompaniment of less combustion. As we have already seen, he used these data to support a particular theory of embryonic growth (see Section 2'6), but it is sure that the problem cannot yet be regarded as settled especially in view of the fact that many researches demonstrate the energetic efficiency to be uninfluenced by temperature (cf. the Section on energetics)^.
The P.E.C. of embryonic growth would seem to be distinctly higher than that of post-embryonic growth. An example could be taken from the work of Farkas and Kellner on the silkworm. On the other hand, if only the early part of development in some organisms be considered the P.E.C. may be still higher. Thus Parnas & Krasinska calculated that as a hatched frog larva might be regarded as containing 44 per cent, dry solid, of which about 1 2 per cent, would be unused yolk, 32 per cent, would be the dry solid of the embryonic body. Now one frog's egg, according to their data, consumes 27 c.mm. oxygen from fertilisation to hatching, or 0-039
1 The work of Wood furnishes a suggestion with regard to this discrepancy. He found that trout embryos reared at 7° and 12° gave a P.E.C. of 0-63, which was not far from Gray's figures for 10°. At 3°, however, the P.E.C. was 0-55. In Wood's view, constancy of P.E.C. only occurs within the optimum range of development, and at lower or higher temperatures the processes of combustion and storage are dislocated. But, paradoxically, Wood's final larval size was smaller the lower the temperature ; exactly opposite to the results of Gray.
SECT 6] OF THE EMBRYO 939
mgm. which would correspond at the outside to 0-054 mgm. carbon dioxide or 0-0146 mgm. carbon. And the hatched larva weighing 2 mgm. has about 0-6 mgm. of protein in it, or 0-3 mg. carbon, so that only about 5 per cent, of the carbon absorbed was burned. The P.E.C. was therefore about 95 per cent, or 0-95 but of course hatching in amphibia is not the end of development, and before all the remaining yolk is used up the efficiency will have fallen to the usual 60 per cent, or 0-6.
6-1 1. Metabolism of the Avian Spare Yolk
This will be a convenient place in which to give an account of the yolk which is still available for the chick at the time of hatching. In the succeeding sections of the book, the metabolism of the yolk and white during the pre-natal stages within the egg will be fully unravelled, in so far as this is possible in the present state of our knowledge, and the data will be found in their appropriate places according to the section-headings. But the "spare yolk", as it is called, has been investigated so little, and its significance so seldom discussed, that it may as well be dealt with here. The most complete study of it is that of Iljin, though it was first attacked by Virchow. Iljin allowed newly hatched chicks to go without food for a number of days and from time to time weighed the bodies and the unutilised spare yolk. His data, which are plotted in Fig. 255 show that although the dry weight of the yolk diminishes enormously during the first few days after hatching, the dry weight of the chick remains practically constant. This yields an important result, namely, that the chick has absorbed all that it required for architectural purposes from the yolk before it hatches, and that after that moment, the "spare yolk" plays an entirely nutritive part, functioning mainly if not entirely as combustible material. "The chicken Hves on its yolk", said Iljin, "and does not destroy the organised parts of its body, which have only just been formed." Now as will appear in later sections, and largely owing to the work of Riddle, we know that during the last week of incubation, the chick absorbs material from the yolk in varying intensity, lipoids being assimilated more rapidly than fats, and neutral fat more rapidly than proteins. It looks, therefore, as if the yolk at the time of hatching is much more preponderantly composed of protein than it was at the beginning of incubation, and as we know from Iljin's experiments
940
GENERAL METABOLISM
[PT, III
Spare ""
Yolk Chick • O Faverolle] B D Gudan MLjin
that after hatching it is entirely used for purposes of combustion, we cannot avoid the conclusion that the catabolism of fatty acids which was so prominent a feature of the pre-natal period, gives place to combustions in which protein plays a greater part, as soon as hatching is completed. Nor is it unwarranted to see in this arrangement a state of affairs quite in accord with the fact that disposal of nitrogenous waste-products is not easy before hatching and is easy afterwards. For further discussion on this subject see Section 9-15.
Referring again to Fig. 255 it will be noticed that if the chicks were given food a day or two after hatching, there was hardly any diminution in the dry weight of the spare yolk, as is indicated by the dotted line. No doubt it takes a long time to disappear completely if the circumstances of post-natal life are favourable to survival. "Extracting the spare yolk from the stomach", said Iljin, "we saw that it is included in a special tunicle like a sack. This sack opens into the thin gut by means of a special connection, the inside surface of which is corrugated, like the bile-duct of man. The wall of the gut makes a wrinkle above the opening which covers it like a valve, so that the contents of the gut cannot enter the duct during the peristaltic movements."
That the spare yolk forms a considerable part by weight^ of the newly hatched chick is clear, in any case, from Iljin's data, which show from 13-6 to 27-8 per cent, of the wet weight and from 23-1 to 52-2 per cent, of the dry weight.
Schillings Bleecker
Hatclning
Fig- 255
^ The exact degree to which yolk is assimilated prior to hatching has been investigated by Jull & Heywang. There is no sex difference in the amount of spare yolk, but considerable variation according to the hen laying the eggs. The average amount of spare yolk is 40-78 % of the original yolk, but it may vary from 32*14 to 46-88 %, and as the eggs from individual hens are fairly uniform in this respect, the phenomenon appears to be genetic in origin (cf. the teleostean hybrids described on p. 920).
SECT. 6] OF THE EMBRYO 941
Later work by Schilling & Bleecker afforded further data about the absorption of the spare yolk by the hatched chick. They observed sometimes a curious failure to absorb it; thus in one instance an amount of 4-8 gm. was found when there should, according to the normal curve, only have been 0-021 gm., and in another case 2-95 instead of 0-62. Their conclusions differed a good deal from Iljin's, for they found no difference in rate of absorption between well-fed and ill-fed chicks^, nor did the amount of yolk unabsorbed seem to bear any relation to the growth-rate of the individual.
Romenski, a student of Iljin's, studied the question from another angle, that of nitrogen utilisation, and drew up, as the result of his observations, the following table :
Gm. 37-60
30-00
0-499 6-951
Average weight of the chick at hatching
Average weight of chick minus its spare yolk ...
Nitrogen content of chick minus spare yolk (i.e. 57-97 % of the
original store of nitrogen in the egg) ... Average weight of the spare yolk Nitrogen content of the spare yolk (i.e. 32-32 % of the origina
store of nitrogen in the egg) Average weight of shell, membranes, and excreta Nitrogen content of shell, membranes, and excreta (i.e. 9-55 %
of the original store of nitrogen in the egg) ... ... ... 0-058
He then subjected the hatched chicks to 36 hours' starvation or, more strictly speaking, he allowed them no other nourishment than that contained in their yolk-sacs. The results of similar estimations at the end of that time were as follows :
Gm.
Average weight of chick after 36 hours (minus its spare yolk)... 33'6o
Nitrogen content of chick body ... ... ... ... ... 0-534
Average weight of spare yolk after 36 hours ... ... ... S'^S
Nitrogen content of spare yolk after 36 hours ... ... ... o-iii
From these facts it is clear that during the post-hatching period the yolk lost 160 mgm. of nitrogen and the chick's body gained 35 mgm. so the loss by oxidation was 125 mgm. Evidently the original contention of Iljin, that the spare yolk is used much more for energy than for storage, received confirmation through the work of Romenski, and the efficiency would here be extremely low, about 20 per cent. Romenski' s figures permit us to compute what intensity of protein combustion goes on under these conditions. The 125 mgm. of nitrogen
^ Later work by Roberts ; Parker ; Holmes, Halpin & Beach, and Heywang & Jull, agrees with Schilling & Bleecker's view on this point.
942 GENERAL METABOLISM [pt. hi
disappearing may be regarded as wholly protein nitrogen, and will therefore correspond to 783 mgm, of protein. Now the maximum intensity of protein combustion within the egg is 80 mgm. per 100 gm. wet weight of embryo per day, and here we have 783 mgm. per 30 gm. wet weight of embryo per i| days, or 1740 mgm. per 100 gm. per day. There seems little doubt but that in early post-natal life a utilisation of protein can go on which does not seem to be possible at any earlier stage although the protein is there. This fact fits in remarkably with a number of others and leads to certain speculations of much interest, which will be brought forward in Section 9-15.
Table 115.
Uric acid ex
cretion per
Protein
100 gm. wet
catabolism in
weight of
% of the total
body per day
material
(mgm.)
catabolised
Investigator
Adult mammalian liver {in vitro)
—
4-27
Singer & Poppelbaum
Adult hen
(a) Inanition after corn diet
50-100
10-14
Von Knierem ; Schimansk
(b) During corn diet
50-100
6
and Voltz
Newborn chick
Inanition, except for yolk ..
127
5-6
Fridericia and Bohr & Hasselbalch
Chick embryo
14th to 17th day of incubation —
5-6
Fridericia
Throughout incubation
—
2-3
„
It also raises the question of what relation exists between the combustion of protein by the chick in the egg and by the adult hen. It occurred to Fridericia to make a comparison of this sort. Referring to the work of von Knierem and of Schimanski he calculated that the adult hen on an ordinary corn diet, or in a fasting period after such a diet, would excrete an average amount of 0-5 to i-o mgm. of uric acid per gm. body- weight per day. This would be 100 mgm. of uric acid per 100 gm. body- weight per day or 206 mgm. of protein catabolised per 100 gm. body-weight per day, i.e. more than twice as much as the highest point of pre-natal protein catabolism. Fridericia collected the excrement of newly hatched chicks for a day or two after birth, and did not find such large amounts of uric acid as would be expected from Romenski's figures. He found 53*2 mgm. of uric acid per kilo per hour, i.e. 127-5 nigm. uric acid per 100 mgm. per day corresponding to 266 mgm. of protein combusted per 100 gm.
SECT. 6] OF THE EMBRYO 943
per day, instead of 1 740 mgm. However, this was a good deal more than the highest point reached in pre-natal life. Fridericia did not work out the percentage participation of protein in the total combusted material by actual measurements of the fat and carbohydrate disappearing, as was done in later work (cf. p. 1 1 34) but knowing the total heat output from Bohr & Hasselbalch's figures, and knowing the amount of uric acid produced, and hence the amount of protein catabolism and its heat, he calculated the latter in per cent, of the former. Thus he was able to draw up the above table. It must be remembered that the above data for the chick embryo were Fridericia's own, and that other workers on uric acid formation in the egg do not wholly confirm them, but nevertheless, a comparison with these (Table 141) will show that the main conclusions to be drawn are not affected by these discrepancies. The most probable alteration which should be made in the table is a lowering of the last value. Further researches should be undertaken to decide the point at issue between Fridericia and Romenski.
6*12. Maternal Diet and Embryonic Constitution
On p. 248 I discussed the extent to which changes in the diet of the hen could influence the chemical constitution of the egg and thence perhaps of the embryo. In the case of the mammal it is obviously impossible to trace the effects of the diet upon the constitution of the egg, but something has been done on its effects on the constitution of the embryos at birth.
Reeb studied the effects of under-nutrition in rabbits upon their offspring and found that the experimental rabbits produced embryos 41-2 per cent, lighter than the controls, containing 44 per cent, less dry solid, and 61-9 per cent, less fat. The same results emerged, with only minor differences, from work with dogs. Paton's guinea-pigs gave the following results :
Weight of young Average no.
per gm. of mother of young
at term (gm.) per litter
Well-fed ... ... 0-350 2-7
Under-fed ... ... 0-248 2-5
Zuntz and Bondi worked with rats — the former confirmed Reeb, and the latter reported that a rich fat diet caused an unusually high fat content of the embryos. On cows, Eckles observed no effect of
944 GENERAL METABOLISM [pt. iii
moderate underfeeding. As for man, Prochovnik in 1889, on clinical grounds, affirmed that the infants of women on restricted diet were smaller and lighter than those of women on liberal diet. During the European War of 19 14-19 18, which unfortunately provided the German workers with many opportunities of studying this problem, Prochovnik's views were not substantiated. Peller, it is true, found a slight difference in weight, but Momm; Sorgel; Ruge and many others could not obtain any evidence for it. Two considerations, advanced by Zuntz, explain why these results differ from those of the animal researches — (i) in spite of the war diets, the deficiency was not enormously great, and (2) the birth-weight in man is a much less percentage of the maternal weight than in other mammals (see p. 475).
Dibbelt put pregnant dogs on a calcium-poor diet, and found that the calcium content of the embryos at term was normal. Zuntz's work on rats included an experiment of this kind, which gave equally negative results. But on the other hand Fetzer found that iron-rich diets increased the iron-content of the foetuses, while iron-poor ones, if below a certain level, led to abortion and loss of the litter. The effects of vitamine deficiency and excess on the embryos have also been studied, but for an account of the results obtained reference must be made to Section 16-5.
The strain on the maternal organism of providing material for foetal growth is strictly outside the scope of this book, but a word or two on the subject may be said here. Gowen investigated the extent to which the milk yield of cows is reduced by gestation, and after a close analysis of extensive statistics concluded that a cow in the 9 months of pregnancy produces 342 to 628 lb. of milk less than a non-pregnant one. There is thus a 5 per cent, diversion of milk products to the foetus, a diversion, moreover, which would seem to bear equally on all the constituents of the milk, for the butterfat percentage, for example, is not influenced at all. Very similar results were obtained by Brody, Ragsdale & Turner, who found a diversion to the foetus of 450 lb. of milk, i.e. rather more in dry weight than the foetus at birth which is equivalent to 275 lb. of milk. In mice, according to Kirkham, the increased gestation time of nursing animals seems to be due rather to abnormalities of implantation than to any drain on the maternal body.
For the considerable literature on the effect of alcohol and other drugs on birth weight, reference may be made to the papers of Hanson & Heys.
In this connection it is interesting to find general agreement existing that the larger the litter in a mammal the smaller each individual is. Thus Bluhm found the following figures in mice:
No. of embryos Weight in gm. of in litter each individual
And the same holds true for the guinea-pig (Ibsen & Ibsen), the rabbit (Kopec) and the pig (Hammond).
Cite this page: Hill, M.A. (2024, April 28) Embryology Book - Chemical embryology 2-6 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-6_(1900)
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