Book - Chemical embryology 2-4 (1900)
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Needham J. Chemical Embryology Vol. 2. (1900)
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Chemical Embryology - Volume Two
Section 4 The Respiration And Heat-Production Of The Embryo
4-1. Early Work on Embryonic Respiration
Probably the earliest examination of the respiration of embryos, apart from mere opinions such as those of Fabricius ab Aquapendente, was contained in the work of Spallanzani, who found that eggs gave off and took in gases, although Robert Boyle in his Continuation of New Experiments physicomechanical touching the Spring and Weight of the Aire & their Effects had written in 1632, " I put Flies' Egs into an empty receiver: no wormes were produc'd out of them". Nothing more of importance was done till Coxe on May 19, 1794, presented to the Academy of Sciences in Philadelphia An inaugural Essay on Inflammation, in which he stated that "the portion of air which we always find in one end of the hen's &gg is oxygenous gas". His essay is now rare, so I have not been able to ascertain whether he regarded the contents of the air-space as pure oxygen or as simply containing pure oxygen among its constituents. Coxe thought that the air-space was of great importance for the proper growth of the embryo. Two years later, Hehl at Tubingen carried out similar work, using Fontana's modification of Priestley's eudiometer. He concluded that the air was the same as ordinary air.
In 1 8 1 1 Paris made an examination of the physiology of the fowl's tgg, which he communicated to the Linnean Society in London. According to his analyses, the air-space of unincubated eggs contained "pure atmospherical air", but after a development of three weeks there was "an inquination with carbonic acid". For his time, Paris held very advanced views about gaseous exchange in animals — "Is it not probable", he said, "that the repeated suspirations of the fatigued are instinctive exertions to procure a greater proportion of oxygen by which their muscular energy may be revived?" The eggwhite, in Paris' view, was merely a defence against the cold. Then in 1822 Sir Everard Home made similar investigations, in the course of which he submerged eggs in water and other liquids, and observed that they would not develop, a result which he attributed to the absence of the air. It is most interesting to note that none of these early workers seemed to find any difficulty in the notion of a nonpulmonary non-circulatory respiration. In 1840 Bucknell commented on the increase in size of the air-space during incubation.
1 Note: I mgm. COj = 0-51 c.c; i mgm. O.^ = 0-70 c.c. (at n.t.p.).
Most of the attention given to embryonic respiration during the earlier half of the last century was centred on the bird's egg, but a few experiments were done on other eggs. In 1846 the Academy of Sciences in Paris offered a prize for a memoir in which the candidates were required to "determine by the aid of precise experiments what is the succession of chemical, physical, and organic changes which take place in the egg during the course of the development of the foetus in birds and batrachians". The prize was won by Baudrimont & Martin de St Ange, who produced a work which must be regarded as one of the classics of chemical embryology. I shall refer later to their general results on the metabolic changes which they investigated; it is only necessary to note here that they proved that carbon dioxide was given off throughout incubation by the developing eggs of hens, garden snails, lizards, snakes and frogs. They also measured the daily loss in weight of developing hen's eggs, and did not fail to note that this could be at least doubled by incubating the eggs in an atmosphere which had been dried by sulphuric acid. On the other hand, they affirmed that nitrogen was lost by the eggs, and that an egg weighing 50 gm. would give off 0-055 S"^- of sulphur in 21 days, presumably in the form of hydrogen sulphide. They tried incubating eggs in oxygen, hydrogen and carbon dioxide, observing in each case the teratological results, and analysing the gases in the air-space. Frog's eggs placed in a vacuum were found not to have developed at all. Other points investigated by these workers were the permeability of the frog's egg to strychnine and to morphine. But, for the present purpose, it is only to be noted that they initiated the quantitative work on embryonic respiration.
Curious experiments were also made at about the same time by Rusconi and by Preyer, in which larval amphibia were raised in the absence of atmospheric air, simply by the dissolved oxygen in the circulating water, but they are not now of importance. More interesting was the gasometric work of Bischof and of Dulk, who in 1823 and 1830 respectively, without knowing of Coxe's work, analysed the gas which could be extracted from the whole egg when placed in deaerated water in a vacuum. They both found it to have a higher oxygen tension than ordinary air. Von Baer, who was at that time writing the introductory parts of his immortal book on the embryology of the chick, was at once interested, for, just as Haldane a century later was to welcome the secretory theory of the pulmonary epithelium as a basis for views of a vitalistic character, so von Baer saw in the analyses of Bischof and Dulk a like support for his general opinions. On p. 37 he considered the gases of the egg, and, referring to a figure of 27 per cent, of oxygen (atmospheric air 20-5-2 i-o), said, "This value was Dr Bulk's. Previously Dr Bischof paid attention to the air in the egg and found amounts of from 22 to 25 per cent. Although his paper was rather short, I hope Dr Dulk will repeat his observations. The result of this research is for embryology and the whole of physiology so important that I feel it my duty to make very well known these valuable communications". Nothing, however, has been done on the problem since that time. But in 1847 Baudrimont & Martin de St Ange examined the air in the air-space, though it must be admitted that their analytical figures showed as often as not less oxygen in the air-space, not more, than in atmospheric air.
The most surprising feature of the early work, however, was the fact that a number of workers seriously suggested that the hen's egg was quite independent of air during its development. The main upholders of this doctrine were Erman; Viborg and Towne. Reaumur in the eighteenth century had already found (see p. 198) that development would not go on if the egg was covered with some substance impermeable to air, but Erman and Viborg, using gypsum, got different results and affirmed that air was quite unnecessary, for they could hatch out chicks from eggs buried in this way. Towne came to the same conclusion from experiments in which he gummed pieces of paper all over the outside of the shell. Von Baer, as one of his footnotes shows, was prepared to accept a good deal of this work, for it fitted in with his own idea that there was some essential difference between pre-natal and post-natal development. He anticipated, it would almost seem, the discovery of two quite different sorts of metabolism. In 1834, however, Theodor Schwann, destined to be known later on as the first cytologist, proved clearly that air was essential for development, by maintaining eggs in an atmosphere of hydrogen. His inaugural thesis at Berlin, De necessitate aeris atmosphaerici ad evolutionem pulli in ovo incubato, finally settled the question, though for some time other workers, such as Marshall, thought it worth while to make further experiments (e.g. submersion in oil) in support of Schwann's conclusions^.
A good deal of work, however, continued to be done on the effects produced by partial and total varnishing of the egg-shell, and attempts were made to find out just how much of the surface was necessary for satisfactory development. Geoffroy de St Hilaire; Baudrimont & Martin de St Ange; Herholdt; Poselger, and Dareste used varnish, collodion and wax respectively for this purpose, but, as they themselves owned, such eggs lost a good deal of weight during incubation, so that their varnishes must have been permeable to some extent to gases. This explains why in their experiments the allantoic vessels grew quite as usual under the shell. Dareste made a great many observations on these points, but he was not very successful in clearing them up. He stated that, if the part of the shell which covered the air-space was varnished, the allantois would grow over the inner parts of the shell, but not over the membrane separating the egg-contents from the air-space. If the blunt end was varnished before the fifth day, the embryos inevitably died, but if after that time, they did not, for the allantoic vessels had then had time to apply themselves to other parts of the egg's surface. Varnishing the pointed end never had any ill effects. The idea thus grew up (without any real justification) that the air-space had some special significance for embryonic respiration, or at least, that the allantois normally reached the air-space membrane first of all and so made use of its air to a special extent. Dareste also affirmed that, after the varnishing of the obtuse end, the air-space often moved round to the side of the egg. Dusing, who made a thorough examination of the whole subject at Preyer's instigation, was not able to agree with all the conclusions of Dareste. He used an asphaltic preparation which really was impermeable to gases, and carefully ascertained that the eggs varnished all over with it lost hardly any weight during their 3 weeks' incubation. By this means he found that the varnishing of the blunt end of the egg did not lead to a high mortality among the embryos, no matter when it was done, from which he concluded that the allantois does not normally reach the air-space membrane first of all, and that the embryo does not depend upon the air there for the oxygenation of the blood in its vessels. He was able to hatch out a large number of eggs which had been varnished over the air-space in this way. He could not confirm Dareste's statement that the air-space could move round to one side after varnishing.
1 Asphyxiation of insect embryos while still in their eggs has now become a very important part of economic entomology (see Staniland, Tutin & Wilson) .
Busing then proceeded to ascertain exactly how much of the shell surface was requisite for full oxygenation of the embryo. By varnishing little squares in chessboard formation of exactly equal area all over the egg, he found that certainly 50 per cent, of the shell could be occluded without any ill results being produced. In one case, perfectly normal development followed the occlusion of 65 to 70 per cent, of the total surface in this way, but the number of abnormal embryos rose rather rapidly at this point. Gerlach & Koch went even further, and varnished eggs over the entire surface save for a small circle from 4 to 6 mm. in diameter as near as they could judge immediately over the germinal disc. The embryos produced in eggs so treated were often abnormal, but seemed occasionally to be well developed ; in all cases, however, they were much smaller and lighter than the normal. The nearer the "Luftfleck" was to the embryo, the more normal the development. Preyer drew from these experiments the conclusion that a proper supply of oxygen must be more essential for growth than for differentiation, but he did not follow out that interesting line of thought, which has affinities with Demoor's finding that irrespirable gases stop cytoplasmic streaming in Tradescantia, but not nuclear division (see also p. 542). Gerlach, who studied in morphological detail the embryos resulting from this method, found that the abnormalities must in many cases have arisen during the first 15 hours of development, a fact which demonstrated that, even in those early stages, oxygen was necessary. Varnishing experiments were afterwards continued by Fere and by Mitrophanov and low-pressure work by Giacomini. For the complicated early history of the work on the respiration of eggs, with all its details, Dareste's 1861 paper should be consulted.
Baudrimont & Martin de St Ange made one experiment of much interest, in view of the later work of Riddle (see Section 18-9), in which they maintained eggs at 37° in an atmosphere of 85 per cent, oxygen. They found on opening the eggs after some days that the embryo had a red colour, the allantois was a millimetre thick and very resistant, and the amniotic liquid was red, owing to the presence of numbers of erythrocytes in it. Pott & Preyer later repeated this experiment, and found that the description of the earlier workers had been quite correct, only that no blood was to be seen in the amniotic liquid. Excess of oxygen is undoubtedly as deranging a condition as lack of it.
Pott & Preyer also investigated the normal behaviour of the airspace during development, studying its gradual enlargement and its position. They analysed the air in the air-space and naturally found carbon dioxide to be present. This latter point was contested by Berthelot, on the basis of very poor technique, so it was not surprising that Hiifner later was able to agree in full with the work of Pott & Preyer. Preyer made an attempt to explain the early figures of Dulk, etc. by Graham's atmolysis laws, thinking that the shell might be more permeable to oxygen than to nitrogen. But this matter was taken up by Hiifner, whose paper has already been referred to (p. 264), who showed that, on the contrary, nitrogen diffuses through the egg-shell more easily than oxygen.
Quantitative estimations on the loss of weight from the egg during its incubation were made very early by Sacc; Prevost & Dumas; Pfeil; Robinet (on the silkworm) and later by Prevost & Morin. "The diminution in weight", said these latter authors, "which the egg undergoes during the course of incubation, cannot be explained as a simple evaporation. It must be admitted that at the same time as the fatty substances are assimilated or destroyed, a part of the azotic bodies are too, and that there goes on in the egg an act perhaps analogous to respiration, the result of which is the exhalation of such substances as can take on the gaseous condition. The appearance of the membrane which carpets the shell seems to confirm this opinion." The simple fact of loss of weight had been known already for some time ; thus Reaumur in the eighteenth century had observed a loss of 16 per cent., Copineau 14 per cent., Chevreul 1 7 per cent., Prout 16 per cent., Sacc 1 7 per cent. Falck was probably the first to make any measurements of the rate of weight loss each day, but he did not pubhsh many weighings, and Pott & Preyer made a definite advance by increasing the number of eggs under investigation and by paying greater attention to details, such as maintenance of accurately controlled temperature and humidity. Nevertheless, their tables show great variations, and their work is not sufficiently satisfactory to be included in any calculations of importance at the present time. It is true^ however, to say that the main features of the gaseous exchange of the egg were correctly sketched out by Pott & Preyer. Thus they found that the fertiHsed egg developing with the embryo inside it lost on an average 19-6 per cent, of its weight, and that, if the egg was infertile and was yet incubated, it lost nearly as much (18-5), the difference being about I per cent. This coincides with the fact now definitely known that the main source of weight loss in incubating eggs is the evaporation of water, not more than 2 or 3 gm. of solid being burned away. The earliest observer to note that the weight loss of fertile and infertile eggs was much the same appears to have been Erman, who announced it in 1810 in a letter to Oken. Pott & Preyer also made the correct observation that the loss of weight during the incubation period was constant for each day. This, of course, made it obvious that the weight loss was not directly connected with the embryonic growth, the course of which was known by them to be curvilinear. Fig. 105 is a modification of the illustration they gave of their findings.
Fig. 105.
The weight of water W, they said, evaporated each day by the egg as far as the end of the second week, is equal to the total loss of weight, G, for during this time the weight of carbon dioxide produced, K, is exactly equivalent to the weight of oxygen absorbed, S — other gases being omitted on account of their small quantity. Thus
G = r+ W-S and G^W if K ^ S.
Roughly speaking, these relationships are still true.
The question of whether any other gases were given off or taken in during the incubation period by the egg was also handled by Pott & Preyer. Schwann had found that not only carbon dioxide, but also hydrogen and nitrogen, were given off, a result which neither Baumgartner nor Pott & Preyer could confirm. The work which was done later on this point, and which proved that carbon dioxide is the only gas evolved by the developing egg, will be referred to presently.
"The amount of carbonic acid gas", said Pott & Preyer, "given off by a developing embryo in a six-hour period, was four times as great at the beginning of the third week as it had been at the beginning of the second week, and on the twentieth day it was nearly ten times as great as at the end of the first week. During the course of the second week the carbonic acid gas exhaled is more than doubled and during the course of the third week it is more than doubled again." Pott & Preyer rightly felt it to be a very important finding that the embryo used up oxygen and gave off carbon dioxide long before the establishment of a pulmonary mechanism. This work would seem to be of importance in the history of the progress of the conception of tissue respiration, but no one has yet accorded such a credit to it. The absolute values which Pott & Preyer obtained for the amounts of oxygen and carbon dioxide concerned, resemble fairly closely the figures of later workers. By performing a simple calculation
where K^ and K^ is the loss of carbon dioxide by fertile and infertile eggs respectively, W^ and Wy_ the loss of water by fertile and infertile eggs respectively, and Gg and G^ the loss of total weight by fertile and infertile eggs respectively, Pott & Preyer calculated the amounts of oxygen actually used up by the embryo each day. They did not make any direct estimations of oxygen. One of their conclusions was not supported by later experiments, for they said that the loss of water was markedly affected by the presence of the embryo, affirming that fertile eggs lost much less per day than infertile ones. This statement cannot now be accepted without modification.
Although the greater part of our knowledge of the respiration of the mammalian embryo is derived from researches undertaken at a comparatively early period in the last century, the consideration of it will be deferred until the discussion of that subject. It need only be said here that Girtanner and Schehl in 1 794 seem to have been the first workers to say definitely that the mammalian foetus "asphyxiates itself if it does not receive oxygen from the blood of the mother". Experiments by Scheel in 1798 led to the same conclusion. Runge & Schmidtt and Zweifel observed the presence of oxyhaemoglobin in the umbilical vessels.
Before proceeding to the investigations of more recent times, we may mention a few more of the older ones which have a special interest. Audouin; Baumgartner; and Prevost & Dumas all concluded that about three litres of carbon dioxide were lost during the incubation period by one hen's egg of approximately 50 gm. It is striking that, although Audouin's work, for example, was done in 1827, their value should have been so correct, for Bohr & Hasselbalch 72 years later obtained a figure of 3-032 litres. It is also interesting that the values which Pott and Nessler obtained for weight loss come very close to those of twentieth-century workers.
Early work on heat-production was, of course, much rarer. Barensprung in 1 85 1 ascertained by the use of a thermometer that hen's eggs in course of development were one-tenth of a degree hotter than the circumambient air, and Ruffini made a similar observation on toad's eggs. Murray had claimed as early as 1826 that the albumen at the blunt end was a degree or two hotter than that at the pointed end. In 1872 Moitessier compared the rate of cooling of fertile and infertile hen's eggs and found that the latter cooled more irregularly, perhaps because of the allantoic circulation.
4-2. Respiration of Echinoderm Embryos in General
Among the eggs of aquatic animals those of echinoderms have been much investigated with regard to their respiration and heat production, and they have a good deal to tell us about the gaseous exchange of the embryo. The first paper on the subject was that of Lyon, who stated that he had observed a rhythmically increasing and decreasing production of carbon dioxide during cleavage stages in Arbacia eggs, but as he pubUshed no figures in support of this affirmation his paper did not provoke much interest. The first work of importance was done by Warburg in 1908. Using the eggs of Arbacia pustulosa, he determined the oxygen consumed in a given time and at different temperatures by the Winkler method. Instead of weighing the eggs, he introduced the method of doing Kjeldahl nitrogen estimations on them, and of referring the oxygen consumed per hour to these figures, instead of to weight, on the assumption that the total protein content of the eggs remains constant through all the early stages. This was subsequently justified by Rapkine, who measured the wet and dry weights of sea-urchin's eggs. Warburg paid careful attention in this work to the possible errors introduced by bacteria, spermatozoa, and teratological complications. His principal figures were as follows:
Mgm. oxygen used per hour by
weight of egg corresponding
to 28 mgm. nitrogen
Unfertilised egg 0-055
Fertilised egg ... ... ... 0-303
8-cell stage 0-355
32-cell stage ... ... ... 0-576
Warburg mentioned the fact that Loeb had found that unfertilised echinoderm eggs would live well for a week in sterilised water. During this period they would absorb oxygen, so evidently a definite metabolic turnover was proceeding in them. Fertilisation led to a multiplication of the metabolic rate by 6 or 7. Later development seemed to show no change at the 8-cell stage, but some increase at the 32-cell stage. Warburg also investigated the effect of hindering or stopping altogether the cleavage of the eggs by placing them in hypertonic sea water ( I gm.NaCl added to looc.c. sea water). The respiratory rate was practically unaffected, being 0-368 mgm. oxygen per hour per 28 mgm. nitrogen in the normal case and 0-347 i^igm. in the inhibited case. A comparison between the rate of respiration of the egg and the spermatozoon demonstrated that the former respired about 500 times as much as the latter. The influence of hypertonic sea water, however, was found by Warburg to be a little different in the unfertilised egg. Recently Runnstrom has observed an increase of respiratory rate in hypertonic solutions. Loeb had shown that hypertonic solutions were active agents only if they contained dissolved oxygen, and had concluded that they had an effect on the respiratory rate. In Warburg's experiments this actually turned out to be the case; for example:
Treatment for half hour Mgms. oxygen used per hour per 28 mgm. gm. sodium chloride per nitrogen after the eggs had been
100 c.c. sea water put back in ordinary water
I 0-085
2-3 + 1-6 c.c. jV/io soda 0-282
4-3 +3-0 c.c. JV/io soda 0-535
Just the same effects were observed on treatment with hypotonic sea water, so it seemed as if some, at any rate, of the agents which would induce artificial parthenogenesis would also induce the high respiratory rate characteristic of the newly fertilised egg. As was to be expected, the temperature coefficient of the respiration was found to be approximately 2, for at 20° the respiratory rate of unfertilised eggs was 0-059 '^g- oxygen, and at 28° it was o-i 18 mg. oxygen. In his second paper, Warburg dealt principally with the effect of hypertonic sea water on eggs unfertilised and fertilised. He had found in the previous paper that, although the respiratory rate was practically unaffected when cleavage was stopped by putting the eggs in hypertonic sea water, they took up much more oxygen than normally on being returned to ordinary sea water. Thus treatment for varying periods with hypertonic sea water would raise the respiratory rate by a good deal. The rate of unfertihsed normal eggs being taken as i , then the rate of fertilised normal eggs was 6 to 7, that of unfertilised eggs after hypertonic sea water 4 to 5 and that of fertilised eggs after hypertonic sea water something like 20. The figures were as follows:
C.c. oxygen used per hr. Unfertilised eggs : per 28 mgm. nitrogen
After 75 min. in hypertonic solution 2-3 % sodium chloride 0-29
„ 105 „ „ 2-3 „ 0-32
,, 60 ,, ,, 2-3 ,, 0-22
Fertilised eggs in ordinary sea water 0-30 Fertilised eggs:
After 30 min. in hypertonic solution 4-3 % sodium chloride I 'go
>» 60 ,, ,, 2-3 ,, 0'7i
It was noticeable that the utilisation of oxygen by the fertilised eggs in hypertonic solution was not equal to that by the fertilised eggs in sea water plus that by the unfertilised eggs in hypertonic solution, but was much greater. The oxygen consumption seemed to remain at a steady level, and not to rise with time, but it was very little affected by temperature, unlike the respiratory rate of normal eggs in normal sea water. More interesting embryologically was the experiment in which Warburg took eggs at different times after fertilisation, and, placing them in hypertonic sea water in order to raise their respiratory rate and get bigger differences, afterwards determined the amount of oxygen consumed by them per hour per 28 mgm. nitrogen. The figures were as follows :
Minutes after fertilisation
Respiratory rate c.c.
oxygen per hour
per 28 mgm. nitrogen
5 17
0-42 0-73 0-62
125
1-07
1'2I 1-36
Time of first cleavage 100 minutes.
Here was a definite appearance of rising metabolic rate.
In his third paper Warburg took the eggs of Strongylocentrotus as his material. By using phenylurethane he showed that the processes of cytoplasmic and nuclear division were not very closely bound up with the respiratory rate, for they might be greatly depressed, and even abohshed aUogether, without any effect on the respiration-rate being perceptible. Thus, after 25 minutes in ordinary sea water, the astropheres are visible, but nothing has happened at all in 1/2000 normal phenylurethane. After 40 minutes, the beginning of the first cleavage is usually present, but in the phenylurethane eggs the astropheres are only just appearing. After 90 minutes, the two blastomeres are usually each beginning to divide, but the phenylurethane eggs have only got as far as the equatorial plate stage. Yet in spite of these profound differences, the oxygen consumption of the two groups of eggs is not markedly different, the inhibition due to the phenylurethane not being more than 20 per cent., as opposed to the 600 per cent, rise on fertilisation. Typical figures were 0-450 c.c. oxygen per hour per 28 mgm. nitrogen for the normal ones and 0-438 c.c. for the urethane ones. "The visible changes in the early developing egg", as Warburg said, "are not conditions of the change in oxygen utilisation after fertilisation. But on the other hand Loeb discovered that oxygenation is a condition of the visible changes, so that those chemical processes, the activity of which we can judge by the amount of oxygen taken in, would seem to underly the morphological ones."
The work of Runnstrom on echinoderm eggs is also very important. He investigated the inhibition of respiration in mixtures of carbon monoxide and oxygen, and found that it was always greater in the case of fertilised or otherwise stimulated eggs than in the case of unfertilised ones. With 96 per cent, carbon monoxide the inhibition was on an average 64 per cent, if the egg was fertilised, but only 5 per cent, if the egg was unfertilised. He linked up these views in a theoretical discussion with the colloidal changes known to take place on fertilisation. In carbon monoxide atmospheres, membrane formation is unimpaired but no rise of respiratory rate takes place. As for potassium cyanide, very much the same results were found as for carbon monoxide, i.e. the inhibition was greatest on the fertilised eggs.
Runnstrom concluded from these facts that the "Atmungsferment " of Warburg (the indophenol oxidase system) is not "saturated", i.e. not fully in contact with its substrates in the unfertilised eggs. Addition of the Rohmann-Spitzer reagents makes the unfertilised eggs respire just as fast as the fertilised ones, and the inhibition by CO is then the same on both; therefore the "Atmungsferment" is just as active in the former as in the latter. Again, methylene blue is reduced anaerobically just as rapidly by the unfertilised eggs as by the fertilised ones, therefore the dehydrase systems are equally active in both. "Not the Atmungsferment, but the relation between Atmungsferment and its substrates, is what is changed on fertilisation" (Runnstrom). For a further discussion of these facts and their significance see p. 867 and the reviews of Keilin and of Dixon.
Runnstrom also studied the effect of urethane. Here the inhibition of the respiration of the fertihsed eggs was accompanied by a stimulation of the respiration of the unfertiHsed ones, so that the two came to about the same level. Runnstrom found spontaneously occurring instances of inhibited cleavage in which the respiration was normal. He noted that the protoplasm of urethane eggs was much more heterogeneous colloidally than that of ordinary ones.
An influence which was found to be much more certain in its action and easier to control than hypertonic sea water was found to be the hydrogen ion concentration of the sea water. Herbst and Loeb had found simultaneously that this factor exercised an important influence on the normal development of the echinoderm egg, and Warburg now studied its influence on the embryonic respiration. The results were as follows :
Respiratory rate
pH
c.c. oxygen, etc.
Cleavage
6-0
0-14
None
8-0
0-39
Normal
II-O
o-8i
None
The more alkaline the sea water the larger the respiratory rate, but not necessarily accompanied by normal development. The influence of the hydrogen ion on the metaboUc rate in the echinoderm embryo was evidently, therefore, not exerted indirectly through the effect on morphological development. After the experiments, the eggs were put back in normal sea water, but the number which reached the larval stage was not great either in the case of the acid ones or the alkaline ones. The effect ofpH on the respiratory rate of Arbacia eggs was afterwards fully studied by Loeb & Wasteneys, and by McClendon & MitcheH.
The question of how the hydrogen ion concentration brought about this effect was also discussed by Warburg. He showed, by staining eggs vitally with neutral red after the manner originally introduced by Loeb, that the internal pR was not affected by a stay in sea water of abnormal pYl. Later experiments by my wife and myself using micro-injection methods (see p. 845) confirmed this observation of Warburg's, and showed that echinoderm eggs could remain for 3 hours in sea water at pH 6-o (normal 8-4) without undergoing any change in the intracellular pH.
Runnstrom in 1929 re-opened the question of the influence of/)H on echinoderm egg respiration. Like Warburg, he found that the more acid the/>H the less intense the respiration; thus at/>H 6-5 the inhibition was 27 per cent, and at pH. 6-1 51 to 58 per cent. This effect was equally shown by fertilised and unfertilised eggs. Runnstrom remarked that the difference between the pH of the cellinterior (6-6, Needham & Needham) and that of the sea (8-4) appears to be necessary for normal respiration.
Another powerful agent which influences the respiration of these eggs is methylene blue, according to the work of Barron. Addition of this dye to the vessels in which the eggs are respiring much increases the rate at which they do so. The effect is shown, indeed, by any reversibly oxidisable dye, and Barron & Hoffmann have studied the action of the rH indicators (see p. 866) on egg-respiration. It depends (a) on the rH of the dye, (b) on the permeability of the cell. If the dye is positive to the cell the effect is maximal, and decreases with increasing negativity, for in order to bring about a raised respiration the rate of reduction of the dye must exceed the normal rate of oxygen-consumption of the cell. Here the dye is acting as an additional "Atmungsferment " and probably oxidising cytochrome. With methylene blue, the effect is not found, according to Runnstrom, in the case of fertilised eggs.
As a result of his experiments Warburg suggested that the toxicity of various salts for echinoderm eggs was due to their effect upon the oxidations going on in them. Thus he found that the poisonous action of sodium chloride solutions could be abolished by adding a trace of sodium cyanide to them, so that an agent which prevented the great rise in respiratory rate acted as a detoxicant. Other examples of neutraUsed effects could be obtained with calcium, magnesium and potassium chlorides, etc., where it was found that the nearer to normal the respiratory rate was kept the less poisonous the solutions were, and the higher percentage of normal developing embryos occurred. From the same standpoint, the influence of extremely small quantities of gold, silver and copper ions was studied. These might raise the respiratory rate by as much as 63 per cent. But their toxic effects were annulled by traces of potassium cyanide, so that, if the increase in respiratory rate was prevented, development would go on normally after the removal of the operating substances. The rise in oxidation intensity produced by ions of the heavy metals was naturally very interesting, in view of the fact that, not long before, traces of silver had been found by Herbst and traces of copper by Delage to be parthenogenetic agents.
Parthenogenetic agents fall into five groups :
1 . Hypertonic solutions (Loeb) .
2. Hydroxyl ions (Loeb).
3. Traces of heavy metals (Herbst; Delage).
4. Fat-soluble acids (Loeb).
5. Fat-soluble substances such as alcohol, benzene, etc. (Hertwig; Herbst; Loeb).
Of these the first three were now found by Warburg to have a strongly stimulatory action on the respiratory rate of the fertilised eggs, while the last two had the opposite effect. Loeb had indeed found that, for the last two, the presence of oxygen was unessential, and that they would produce their parthenogenetic effect anaerobically. "The first group", said Warburg, "act primarily on the oxidations, from which follows a change in the cell-membrane and hence a change in metabolic rate. The second group act primarily on the membrane and not on oxidations." These researches of Warburg led to a long series of papers by various authors, arising out of the antagonism between sodium chloride and cyanide in affecting the respiration intensity of echinoderm eggs. Loeb & Wasteneys in 19 10 went into the subject, and concluded with Warburg that the respiratory rate of eggs stimulated by salt solution was depressed by the cyanide, so that the toxic action of the sodium chloride was averted. But they disputed many of his other statements, and a polemic followed, which must be read in the original, and cannot here be discussed, as it was largely concerned with matters of technique. During the course of it many new facts came to light about the general effects of salt action on protoplasm. Loeb found that the toxic effects of many agents (neutral salts, sugars, hypertonic and hypotonic solutions, chloral hydrate, phenylurethane, chloroform and alcohol) on the fertilised Arbacia ^gg could be prevented by agents such as cyanide, which depressed the intracellular oxidations. That this was not simply due to the suppression of cleavage was apparent from the fact that some of the toxic agents themselves (such as chloralhydrate) depressed cleavage. These questions were dealt with for some further time in the papers of Warburg; Loeb & Wasteneys and Meyerhof. Other workers who have studied the effect of various salts on the respiration of echinoderm eggs are Loucks & de Graff.
More important embryologically were the observations in which Warburg showed that not only phenylurethane would dissociate the respiratory from the morphological process, but also many other agents, such as narcosis with ammonia. The oxidative intensity, as judged by the intake of oxygen, remained unchanged although morphological development might stand quite still. Moreover, the respiratory rate could be raised to very high levels through the action of hydroxyl ions and traces of heavy metals without any morphological development going on at all. Cyanide here took a special position for, penetrating into the cell, it decreased the respiration-rate considerably, and also decreased the development-rate, not producing any deformations, but simply slowing down the normal processes.
Respiratory rate c.c. oxygen per hour per 28 mgm. nitrogen Cleavage
In sea water ... ... ... 0-372 Normal
In JVy 1 00,000 sodium cyanide ... 0-120 Very slow
In JV/io,ooo „ ... 0-072 None at all
Cyanide and temperature were thus the only two agents found by Warburg which equally affected respiratory rate and morphological development.
That the increase in oxygen consumption did not rise parallel with the increase in nuclear material was fully confirmed in Warburg's third paper. The respiration-rate for Strong^locentrotus lividus eggs in the 2-cell stage was 0-438 c.c. oxygen, and in the 64-cell stage 0-612, an increase of only about 1-5 instead of 32 times. A few experiments in which the carbon dioxide output was estimated gave quite similar results; thus in one experiment the respiratory rate for oxygen was 0-20 c.c. of oxygen and o-i8 c.c. of carbon dioxide. Not enough work on these lines was done, however, to lead to any determinations of respiratory quotient. Possible respiratory differences between monospermic and polyspermic eggs were investigated by Warburg, and found to be slight — thus the respiratory rate in one case for monospermic eggs was 0-456 c.c. oxygen and for polyspermic eggs was 0-498, a negligible difference.
Warburg had been led to speculate on the part played by the actual structure of the cells in metabolism by the fact that various agents, especially pH, seemed to affect the respiratory intensity by acting simply on the surface of the cells. He investigated, therefore, the respiration of acetone preparations of staphylococci and yeast cells, contributing the results in a joint paper with Meyerhof, who investigated the eggs of Echinus miliaris. The oxygen consumption of unfertilised eggs pounded up with sand was compared with that of eggs normal and untreated. In the case of unfertilised eggs, the oxygen consumption was not abolished altogether by this pulverisation, although it was definitely decreased. From 0-5 to 1-5 hours after the treatment a half to two-thirds of the original respiratory rate was found, but during the third hour this sank to between a quarter and a third of the original value. Thus the intact eggs would have a rate of 0-053 c.c. oxygen and the pulverised ones a rate of 0-033 ^.c. In the case of the fertilised eggs, this fall after pulverisation was rather more pronounced; as an example:
Respiratory rate c.c. oxygen per hour per 28 mgm. nitrogen
Unfertilised
2-cell stage
4-ceIl stage i6-cell stage
But insufficient experiments were done with the fertihsed eggs to enable this point to be presented with certainty. These, it may be noted, were the first respiration experiments in which manometric methods were used as opposed to the Winkler titration which had previously been universal. The acetone preparations were found to behave rather like the egg-Breis, for, although the oxygen consumption was much less than in the intact normal eggs, it was yet by no means absent. The integrity of the cells was evidently only partially responsible for the oxygen uptake of the original material^. In a later paper Warburg pursued the question still further. He invented a method of cytolysis in which echinoderm eggs were centrifuged rapidly and shaken violently — egg-preparations according to this method not only took up oxygen, but also gave off carbon dioxide. With this
^ Cf. Penrose & Quastel's experiments with bacteria.
Intact eggs
Pulverised eggs
0-41 0-46
0-93 0-89
0-23 0-14 on 0-26
material he showed that the egg-Breis of unfertihsed eggs respired rather more strongly than the intact unfertilised eggs themselves, but that the reverse relation held true of fertiUsed eggs. What was veryimportant was the demonstration that, though there was a difference of 500 to 700 per cent, between the respiratory rates of fertilised and unfertihsed eggs, this difference practically disappeared in the cytolysed centrifuged material, for it possessed apparently the same respiratory rate, no matter whether it came from unfertilised or from fertilised eggs. Sometimes there might be as much as 15 per cent, in favour of the fertilised eggs, but no more. Moreover, if it was true that the spermatozoon influenced the oxidative activity of the egg almost entirely through its effect on the cell-boundary, it should follow that the addition of spermatozoa either intact or cytolysed to the egg-Breis would produce no rise in respiratory rate. This was actually found. Some typical figures on which the above conclusions were founded are given here.
Respiratory rate (c.mm. oxygen used in 20 min. by amount of egg corresponding to 20 mgin. nitrogen at 22°; several experiments)
Unfertilised eggs, intact ... 14 13 12
Unfertilised eggs, destroyed ... 21 22 21
Unfertilised eggs, destroyed ... 25 22 26 23 21
Fertilised eggs, destroyed ... 21 23 25 22 19
Oxygen used up in c.mm.
Brei plus
Brei plus sperm in
Control sperm in distilled
Minutes Brei alone sea water water
20 29 27 30
40 51 50 51
60 67 67 71
In some of the experiments Warburg estimated the carbon dioxide production, getting respiratory quotients of 0-68 and o-8o, but he laid no stress on them for various reasons connected with technique.
Warburg used these " Granulasuspensionen " of the centrifuged cytolysed eggs for the purpose of studying the effect of added iron on the oxygen uptake. It may suffice to say here that he showed that the addition of minimal amounts of iron salts increased greatly the uptake of oxygen by the centrifuged cells. He also showed that it stimulated the production of carbon dioxide by the same material, and that the excess respiration brought about by the addition of iron was of the same type as the normal respiration, judged by behaviour towards ethyl urethane. He tried the effect of adding acids of various kinds and other substances to the Breis, and the inhibiting effect of the cyanide ion was cleared up on the supposition that iron-catalysed oxidations were the main ones taking place in the eggs.
About this time Loeb & Wasteneys undertook an examination of the temperature coefficient of embryonic development in the seaurchin's tgg as related to the temperature coefficient of its respiratory rate. This investigation has already been referred to in the section on growth (p. 525). In another paper they showed that fertilisation in Arbacia eggs led to a three to four times rise in respiratory rate, thus confirming Warburg's work on Strongylocentrotus.
Warburg did not omit to study the effect of varying oxygen tensions on the respiration of his echinoderm eggs. This question, a part only of a very general perplexity which has confused physiologists for many years, he answered by the finding that respiration was relatively unaffected by changes in partial pressure of oxygen. In his 1908 paper, he said, "The oxygen-concentration was so arranged that it did not sink to below f of its original value, but I found that even if it sank to J, absorption proceeded quite regularly". Two years later he said, "I have shown that the rate of oxidation in the egg is independent of the oxygen pressure, i.e. the oxygen concentration in the egg, although this oxidation-rate can be markedly influenced by alterations in various external conditions". Warburg's figures were as follows :
Later, however, Henze went into the matter in a research which included work with anemones, gephyrean worms, and many other marine animals. His conclusion was that Warburg's sea-urchin eggs had not been well enough shaken, for a typical result of his own was
of oxygen
Utilisation
37-4
14-2
3-7
2-1
634
Henze admitted that after a certain point, as was shown later for Dixippus morosus by v. Buddenbrock & v. Rohr, and for Fundulus heteroclitus by Amberson, Mayerson & Scott, the oxygen utiHsation ceased to follow oxygen partial pressure. Subsequent work with other eggs has tended to confirm Henze rather than Warburg ; thus Dakin & Dakin and Burfield have shown for the plaice's egg and Parnas & Krasinska for the frog's egg that the respiratory rate depends directly on the partial pressure of oxygen, below a certain point. Recently Drastich, working with the eggs of Strongylocentrotus lividus, has shown that there is a linear relation between cubic millimetres oxygen used per gram per hour and log. partial pressure. This would imply a state of affairs in which the curve of oxygen consumption fell off rapidly at low partial pressures (see Figs. 106 and 107). And Amberson finds no change in oxygen consumption of Arbacia eggs between 228 and 20 mm. partial pressure of oxygen, though below that it falls off quickly. Everything seems to depend upon the level of oxygen concentration at which this point comes; thus Loeb, in his work on Ctenolabrus eggs, could find no difference in rate of development or morphology between eggs in air or in 100 per cent, oxygen, and Krogh & Johansen successfully hatched plaice eggs in oxygen pressures of one-quarter the normal (230 mm. abs.). Below this point abnormalities occurred.
Fig. 106.
Fig. 107.
Warburg returned to the subject of normal respiration in 19 15, using manometric methods instead of the Winkler titration. He first obtained a more accurate figure for the oxygen utiHsation of spermatozoa, namely, 66 c.mm. oxygen per 20 mgm. nitrogen in 20 minutes at 23°. On the other hand, 20 mgm. egg nitrogen used 10-14 c.mm. oxygen in 20 minutes at 23°. Warburg found that the rise occurring at fertilisation took place within 10 minutes, during which time the respiratory rate rose six times. In the course of further development it continued to rise, so that, after 6 hours, it had reached twelve times the value of the unfertilised egg, after 1 2 hours sixteen times, and after 24 hours twenty- two times. Warburg's curve, which is reproduced in Fig. 108, is very regular, and shows no peaks or rhythmic changes.
Fig. 108.
For the gaseous exchange of the fertiHsed Strong^locentrotus egg^ respiratory quotients closely distributed around o-g were found ^.
1 Values of unity both before and after fertilisation were later reported for echinoderm eggs by Ashbel.
Warburg did not draw any conclusion from this finding, but it has acquired importance in view of subsequent researches on the nature of the substances combusted as energy sources during embryonic development. It will be again referred to in the section on the energy relations of the growing embryo.
The figures given above demonstrated that weight for weight the spermatozoon was respiring more intensely than the egg-cell. When, however, the relations between the respiration of one spermatozoon and one egg-cell were compared, it was found that spermatozoon : unfertilised egg was as i : 500, while spermatozoon : fertilised egg was as i : 3500. Another interesting calculation which Warburg made from his experimental data was that only 0-0045 mgm. of spermatozoon nitrogen were required to fertilise 7 to 8 mgm. of egg nitrogen, i.e. to fertilise i mgm. of egg nitrogen TsW to 20W mgm. spermatozoon nitrogen were necessary. One conclusion from all this was that, as far as respiration experiments were concerned, it was unnecessary to make much correction for the spermatozoal respiration, owing to its extreme smallness.
The next step forward was taken by Shearer, who in 1922, by using a special form of the Barcroft differential manometer, was able to carry out the fertilisation of Echinus microtuberculatus eggs actually inside the closed chamber of the apparatus, and observe more intimately still the earliest stages of the embryonic respiration.
Fig. 109.
Fig. 109, taken from Shearer's paper, shows the phenomena which may under such conditions be observed during the first 10 minutes, i.e. during the period which elapsed between fertilisation and the first readings of Warburg's curve. The two curves show the oxygen consumed and the carbon dioxide in c.mm. given ofT during the fertilisation and early development of an amount of egg-substance corresponding to 4-06 mgm. of nitrogen (approximately half a million eggs) in the case of Echinus microtuberculatus. The respiratory quotient for this experiment was 0-92. The lower line in Fig. 109 shows the oxygen consumption of half a million unfertilised eggs (1-5 c.mm. in 10 minutes). The difference between this figure and the 56 c.mm. of oxygen taken up in the same period immediately after fertilisation is very striking. All the other graphs obtained by Shearer were of the same form, from which it is evident that the uptake of oxygen in the first minute is not only many times more than during any minute before fertilisation, but also more than at any subsequent minute. After the first couple of minutes the rate of increase of metabolic rate falls off, and the curve ascends rather less steeply. The oxygen consumption per unit weight (calculated on nitrogen basis) of the unfertilised eggs per minute was found to be 0-15 c.mm., but the same eggs fertilised consumed in the first minute after the addition of the spermatozoa 12 c.mm. of oxygen, an increase of about eighty times the former value. Shearer compared the respiratory rate of the eggs before and after fertilisation with that of the liver of a well-fed cat (from values in the literature) and obtained the following comparison :
Respiratory rate Cat liver ... ... ... 107
Unfertilised egg ... ... 0-37
Fertilised egg ... ... ... 13-8
Now examination of sections of fixed material of Echinus eggs during the process of fertilisation shows that the spermatozoa take at least lo to 15 minutes to embed themselves in the cytoplasm of the egg. In material fixed within 2 or 3 minutes of the addition of spermatozoa to eggs the former are found only attached to the external surface of the egg-membrane, not having had time to penetrate it. Comparison of these facts, then, with the experimental evidence, makes it clear that the initial burst of oxygen consumption must be brought about simply by the first contact of the sperm with the outside of the egg-membrane. The great rise in metabolic rate which occurs in the very earliest stages of development cannot depend on the formation of the male pronucleus in the cytoplasm, and must be due to some activity exerted by the spermatozoon before it has entered the egg at all. Moreover, when the fusion of the male and female pronucleus takes place later on there is no fresh rise in the process, but rather a further slowing down. This is illustrated by Fig. 1 1 o, which shows an experiment covering a longer period. At the 25-minute point, where the nuclei fuse, there is no kink in the curve, and the process is slowing off. "The nuclear features of syngamy", as Shearer puts it, "seem connected in no direct way with the oxidations taking place in the ovum,"
Such a conclusion is in agreement with the work of Warburg, which, as we have already seen, led to the view that the surface of the egg was of particular importance with regard to the oxidations proceeding in it, and hence the respiratory rate. Injury to the egg-membrane is invariably followed by a great increase of oxygen consumption of echinoderm eggs, and, as will be noted presently, Meyerhof found that similar treatment was accompanied by a definite increase in heat production. In eggs treated with hypotonic sodium chloride solutions, the absence of calcium and potassium ions interferes with the normal condition of the cell- wall, and the oxygen consumption rises to five or six times the normal. In just the same way the heat production rises from o-g to 3-4 gm. cal. per hour after treatment with valerianic acid, which induces artificial membrane formation. There is as yet no satisfactory explanation for these phenomena, for the most recent knowledge which has accumulated about oxidation processes can hardly allow us to be satisfied with the simple conception of an accumulation of iron in the surface layer of the egg, as Warburg suggested. Even if it were there, and were released in some way by parthenogenetic agents, it does not, as is now known, catalyse all types of oxidation. However, Runnstrom's work has shown that the rise in metabolic rate which occurs at fertilisation is connected both with the colloidal state of the cell-interior and with the degree of contact between the "Atmungsferment " and its substrates.
There is no need to suppose that the "Atmungsferment " is concentrated in the cortical layers of the egg, for in a protoplasmic system the transmission of a physical change would readily occur (see also p. 867).
Shearer also carried out some experiments on the glutathione content of the eggs before and after fertilisation. He stated that, before fertilisation, they only gave a weak nitro-prusside reaction, but that, immediately after fertilisation, deep magenta colours could be obtained by this test, indicating the presence of reduced glutathione in some quantity. "There seems to be fairly substantial ground for believing", he said, "that there is an immediate increase in the quantity of this remarkable body in the ovum on fertilisation." Work with the nitroprusside test, however, must be interpreted with caution until quantitative estimations of glutathione have been done on the early stages of the developing echinoderm egg by the iodine method or by some other suitable technique^.
As regards the great initial rise in metabolic rate in the first minute of the experiment, there is every probability that it represents the results of egg-oxidations. But in view of the recent work of Gray, this cannot be said to be a certainty, for Gray, examining the respiration of spermatozoa, finds them to have a much larger and more variable metabolic rate than had been suspected by the earlier workers. Warburg's original figures relating spermatozoa oxygen consumption to egg oxygen consumption were obtained in very concentrated sperm suspensions, and Gray has been able to observe much higher rates of oxidation, especially during that period of activity which the spermatozoa go through just before the eggs are fertilised.
Other and less important measurements have been made of the oxygen consumption of echinoderm eggs before and after fertilisation. Thus McClendon & Mitchell in 19 12, using the Winkler method, demonstrated a rise of six to eight times the previous value at fertilisation, whether natural or parthenogenetic, in the case of Arbacia punctulata. McClendon's theory was that fertilisation led to permeability changes in the eggs which permitted a greater volume of gas to pass in and out per unit time.
- Rapkine's more quantitative experiments indeed show a diminution of SH on fertilisation followed by a rise to the time of first cleavage.
As will be seen later, Meyerhof made a good many estimations of oxygen consumption during the early stages of development of the sea-urchin's egg, in connection with his researches on the calorific quotient and the heat production. He did not devote much attention to analysing his oxygen data, since they were obtained only as a means to an end, but Bialascewicz & Bledovski showed later that they fell on a parabolic curve which was fitted by the equation
x=kfi + a,
where x is the respiratory rate at time t, a the rate in the case of the unfertilised egg, and k a constant. They drew no theoretical conclusion from this, nor did they give the constant in question.
So far all the workers mentioned have been in agreement concerning the effect produced on the oxygen consumption of echinoderm eggs on fertilisation. The first discordant voice was that of Loeb & Wasteneys, who in 1 9 1 2 reported that the eggs of the starfish (species not given) took up no more oxygen after fertilisation than before. The measurements were made by Winkler's method, and the number of experiments described was small, so that the paper did not attract much attention until Faure-Fremiet's similar results with Sabellaria. Later still, Barron & Titelbaum found no rise in respiratory rate on fertihsation in Nereis. The work on the starfish has been confirmed by Barron. That there is a tremendous rise in metabolic rate, however, during the early cleavage stages of most echinoderm eggs cannot now be doubted, although the shape of the curve in the few minutes after fertilisation is still open to revision 1.
Much interest attaches to somerecentexperimentsof Carter in which the effect of the egg-secretions upon the spermatozoa were studied. The known factors here are: (i) Lillie's effect — the agglutination of spermatozoa; (2) Glaser's effect — the lipolytic action (see Section 14-3); and (3) Gray's effect — the increase in oxygen consumption of the spermatozoa. This increase was not found by Carter to follow the same laws as Gray had stated, i.e. there was no falling off of rate (unless the sperm cells were unripe) but on the contrary a constant uptake. This constant uptake was uninfluenced by thyroxin although the falling uptake of unripe cells was increased by that hormone. As iodine is a well-known parthenogenetic agent, there are some possibilities tliat iodine or a thyroxin-like body is contained in egg-secretion.
1 It is interesting to note that increased respiratory rate on fertilisation has been reported for the minnow (Boyd), the silkworm (Ashbel), the tunicate, Ciona intestinalis (Runnstrom), and various amphibians (Bialascewicz & Bledovski; Parnas & Krasinska).
4-3. Rhythms in Respiratory Exchange
From the researches we have so far been discussing no evidence has been found of a rhythmical activity in the gaseous exchange of echinoderm eggs. Lyon's paper of 1904 has been referred to, however. "In nearly all experiments", he said, "there was an increase in CO2 production during the first ten or fifteen minute interval following fertilisation. The increase was slight, and sometimes could not be detected. Following this came an interval in which the GOg production was small, visibly less indeed in two or three experiments than that of the unfertilised eggs and sperm. This is the mid-period of cleavage, approximating perhaps to the time of nuclear growth and the early stages of karyokinesis. The interval during which the eggs were dividing into the first two blastomeres was one of active GOg production. After this period came an interval of lessened production. In one or two cases a second rise occurred at about the time of the second cleavage." And in his second paper on respiration and susceptibility, he said, "It may be stated that the apparent conclusion was that GO2 production in the egg is not uniform throughout the whole series of morphological changes in cell-division, but rather reaches a maximum at the time when the cytoplasm is actively dividing. Furthermore it seemed that at the time when oxygen is most necessary and presumably is being used in largest amount (as indicated by susceptibility to lack of oxygen and to KGN) GO2 is produced in largest amount. If the conclusion above expressed should justify itself it would indicate that oxygen is used chiefly in the egg for synthesis rather than for combustion, and that the larger part of the GO2 comes from spHtting processes. One would also infer that the energy for cell-division comes from fermentative rather than oxidative processes". Obviously these points were of great importance for a knowledge of the metaboHsm of the egg during the earliest stages of its development, and it was unfortunate that Lyon gave no figures in support of his opinions. The theories of cell-division subsequently associated with the names of Robertson, McGlendon, Spek and Heidenhain would then have been easier to assess, for they all postulated the existence of some more or less actively contractile mechanism in the cell during cleavage in contradistinction to the theory of Gray, in which cleavage is essentially due to a rearrangement of the different cell-phases round the growing asters, and in which sudden changes in surface energy of the cell at its equator are not involved. If a contractile mechanism of any sort exists in the dividing cells of the early embryo it would be reasonable to expect some sort of periodicity in the gaseous exchange, just as muscular exertion would be expected to produce it. The sudden activity of the cleaving mechanism would probably be marked by a change in the observable properties of the developing embryo. Lyon's work, then, failed to give an answer to a very important question. In 1922 Vies made an investigation of the problem, using the eggs of Paracentrotus lividus and a quite new method, consisting of the immersion of the developing eggs in a solution of thymolsulphonephthalein, and the observation of the slight colour changes in the indicator by a spectrophotometer. The method was an ingenious one, and the results obtained by it afforded evidence of a rhythm of carbon dioxide production. As the indicator measurements only gave indirect evidence of the formation of acid, a curve with amount of carbon dioxide as one axis could not be drawn, but Vies constructed instead a curve relating p¥L to time. For about 3-5 hours after fertilisation the solution surrounding the eggs became more and more acid, but after that time the acidity rose rapidly, falling off and reaching a Httle plateau just before the completion of the first cleavage. During the 2-cell stage, exactly the same relationships were observed, first of all a rapid rise, and then a falling off to a plateau immediately preceding division. During the stages of 4, 8 and 16 cells, the same cycle was repeated, but the formation of the blastula stopped the rhythmical process, and the eggs then remained for some time causing only a slight change in the tint of the indicator surrounding them. Vies concluded that blastulation involves a change of some kind in the metabolism of the embryo. He recalled in connection with these rhythms the cyclical behaviour noted by Herlant, Heilbrunn and many other workers in susceptibility, viscosity, etc., during early embryonic cleavage (see Section 18).
Gray determined to test the question experimentally by following the cell-divisions through, while estimating the oxygen consumed manometrically. This he did, paying careful attention to the necessary precautions, and obtaining the results shown in Fig. 1 1 1 . The smoothness of the curve is remarkable, and gives no indication at all of any rhythms. Fig. 112, also taken from his paper, shows the result of taking the slope of the curve in Fig. 111 at successive points, and plotting them together. The rate rises clearly from about 2 1 to about 33 per cent, during the 375 minutes elapsing between fertilisation and the end of the 8-cell stage. It is obvious from this graph that the fluctuations in the rate of oxygen consumption bear no relation to the periods of cleavage, but must depend on other variable factors. The curves given by Vies are comparable with the curve for oxygen consumption given by Gray in Fig. 1 1 1 , so that there is a contradiction between the results obtained by the two methods. In such a case, one can only accept the results given by the most direct and accurate method, so that it is necessary to conclude with Gray that there are no rhythms in metabolic intensity during the cleavage of the echinoderm egg. For the details of the criticism of Vies' technique the original papers must be referred to. It should be observed that Vies' rhythms of carbon dioxide production do not agree with those of Lyon, for in the former case the period of maximum carbon dioxide evolution occurs immediately after division, and in the latter case during it. The rhythms of Lyon correspond rather to the rhythms of susceptibiUty to various agents which the echinoderm egg has been found to undergo during its cleavage stages. These will be discussed in the Section on susceptibihty. Having decided, then, that the oxygen consumption gives a better measure of the metabolism in a case such as that of the sea-urchin's egg, there remains the possibility that the rhythms observed by Vies in carbon dioxide evolution may be real phenomena. For, supposing that the carbon dioxide were produced in the cell at a uniform rate just as the oxygen is apparently consumed at a uniform rate, there might still be difficulties in observing this. Nothing is yet known about the alkali reserve of the echinoderm egg, and other factors, such as the alkalinity of the external medium and the surface area of the egg, not accurately controlled in Vies' experiments, might be expected to play a large part in conditioning the results. "It is clear", said Gray, "that since the periodicity in carbon dioxide output is not accompanied by a periodicity in oxygen uptake, the former cannot be regarded as of significance in respect to energy changes in the egg, unless they be due to some obscure form of anaerobic activity for which there is no evidence."
Fig. 112.
The smooth curve obtained by Gray for oxygen consumption of eggs is in agreement with the earlier observations of Warburg, using the Winkler method. It is a very important key position to have gained, for in the light of it various theories which have been proposed from time to time are stripped of some of their attraction. Thus Matthews and Osterhout both claimed that the nucleus played an active part in the oxidative mechanisms of the cell. This is an old idea, and we have already referred to it in connection with the "growth-catalyst". It was the basis of R. S. Lillie's work on intracellular oxidations, and the association of ideas "nucleus-oxidationsgrowth" lies at the bottom of much of Child's writing. Its influence can be traced through region after region of investigation between 1900 and 19 1 8. Gray's work on the oxygen consumption of Echinus eggs demonstrated that, if the nucleus had anything to do with oxidations, its influence must be altogether independent of the phase of nuclear activity, and must be unaffected by the presence or absence of definite nuclear boundary. Again, Robertson in his book suggested that differentiation and growth were dependent on the relative concentration of some catalyst in the nuclear and cytoplasmic portions of cells. This autocatalyst being formed within the nucleus can only enter the cytoplasm when the nuclear membrane breaks down during each prophase. Robertson does not actually say that the autocatalyst is an oxidising agent, but he accepts the view that the presence of the substance can be gauged by the intensity of cell-respiration. On Robertson's theory, therefore, rhythmic changes during early embryonic development in the gaseous exchange would certainly be expected, and it is interesting that, where they can be measured best, no rhythmical phenomena occur. The facts are much more in agreement with the view of Warburg, expressed in his review of 19 14, that the respiration of echinoderm eggs is a function of the cytoplasm, and is independent of the synthesis of the nucleus. Matthews' observation that, in the absence of oxygen, the astral rays disappeared, need not mean that they use oxygen, but simply that, in the absence of the fundamental metabohc processes normally going on, the morphological processes are inhibited. This resembles some of the effects we have already noticed in the work of Warburg.
Fig. 113.
Shearer followed the oxygen consumption of echinoderm eggs for the first lo minutes after fertihsation. Gray as far as about i6 hours, i.e. as far as the i6-cell stage in certain cases, and Warburg as far as 25 hours, i.e. gastrulation. Rapkine in 1927 followed the process further still. Warburg and Shearer referred their determinations to amounts of nitrogen, but Rapkine improved on this by using dry weight. His estimations were done by the Winkler method. The results were rather perplexing, for, when the mgms. of oxygen consumed per I gm. dry weight of eggs were plotted against time (see Fig. 113), a curve was obtained which ascended slowly towards the 24th hour, after which it entered on a plateau indicating apparently a uniform metabolic rate as far as the 40th hour. This in itself agrees fairly well with Warburg's curve, except for a slight initial fall about the 3rd hour after fertilisation, for which there is no explanation. The difficulties which have already been mentioned with respect to Vies' work on the carbon dioxide production naturally operated in Rapkine's work as well, though it was much more satisfactory in so far as the gas was bubbled off, and the carbon dioxide estimated by a baryta method. The alkali reserve, of course, remained an unknown factor. The curve for carbon dioxide given out per hour per gram dry weight followed for the first 9 hours a regular course concave to the abscissa. After that time, however (see Fig. 114), it dropped to a trough at the 15th, and afterwards rose again to a peak at the 24th, after which it again fell and rose to the 40th hour, i.e. to the pluteus stage. The large waves seen in the curve after the loth hour are undoubtedly due to the formation of the skeleton, which is so prominent a feature of the later development of the echinoderm egg As Rapkine & Prenant showed, very definite pH changes take place in the blastocoele cavity as the calcareous spicules are formed and the mesenchyme develops, which are associated with the retention of carbon dioxide to form calcium and magnesium carbonates. Moreover, as Herbst showed, if potassium chloride is added to the water, skeleton formation is inhibited, and in such a case Rapkine found a regularly rising carbon dioxide curve. The respiration curves after the loth hour are therefore difficult to interpret, and attention must rather be directed to the time elapsing before that point of development is reached. During this early period, the metabolic rate, both as regards oxygen and carbon dioxide, is rising, but significantly not at the same rate, so that, when the respiratory quotient is calculated, it begins just after fertilisation at about unity (good agreement here with Shearer and Warburg), but then rises to the extreme value of 4*0 at 4 hours of development, after which it falls away to unity, and remains there (due allowance being made for the influence of calcification) until the pluteus stage is reached (see Fig. 115). Rapkine concluded from this that the enormously high respiratory quotient of the 4th hour indicated a period dominated by syntheses and in which a minimal amount of combustion was proceeding. Coupled oxidation-reduction reactions, the significance of which for embryonic development as a whole will be dealt with in the section on Energetics, would here
Fig. 114.
Fig. 115
be proceeding to a greater extent than simple combustions, so that the carbon dioxide put out would be out of all ordinary relation to the oxygen taken in. Were this the case, the heat given out as actually found calorimetrically would be smaller than that calculated from the absorption of oxygen (endothermic reactions, which catch and hold the heat for the organism, predominating), unlike the state of affairs found to hold for the chick's later stages by Bohr & Hasselbalch. Now Rapkine in a later paper calculated that this was actually the case. Shearer found that, during the first 12 hours of development in the sea-urchin, 25 1 -96 calories were given out, but the heat corresponding to complete combustion of 31-3 mgm. of protein, and 1-65 mgm. of fat (chemical analyses of Rapkine) amounted to 331-3 gm. cal. or half as much again. The discordance between the observed and calculated values was even greater if the figures of Meyerhof for heat production were used, and somewhat less if those of Rogers & Cole were substituted for them, but in no case did the results of the two methods coincide, the difference being at least 40 gm. cal. These facts led Rapkine to the conclusion that in this early period simple combustion was to a great extent complicated by coupled reactions, one member of which was endothermic. He saw in the low calorific quotients of Meyerhof and the preUminary heat absorption phase of Bohr & Hasselbalch still further indications that such processes might be by no means negligible.
4-4. Heat-production and Calorific Quotients of Echinoderm Embryos
The work on the heat-production of echinoderm eggs divides itself roughly into the long papers of Meyerhof in 191 1, the work of Shearer in 1922, and of Rogers & Cole in 1925. Meyerhof made use of a simple apparatus consisting of a Dewar flask immersed in an accurate thermostat together with a Beckmann thermometer, giving readings correct to -001°. With this apparatus he made many experiments on the production of heat by the developing sea-urchin embryos. One of his typical results was as follows :
Heat produced in gm.
cal. per hour per amount of .S" rongylocentrotus lividus eggs corresponding to 1 40 mgm. nitrogen Eggs before fertilisation ... ... ... o-Sg-o-gi
1st hour after fertilisation ... ... 4-0 -4-2
2nd hour (transition to 2-cell) ... ... 4-5 -5-0
3rd hour (4-cell stage) ... ... ... 5-3 -5-8
4th hour (8-cell stage) ... ... ... 6-o -6-5
5th hour (16- to 32-cell stages) ... ... 7-8 -9-5
6th hour (32- to 64-cell stages) ... ... 9-8
14th hour (larvae begin to swim) ... 12-9
1 8th hour 17-8
Another of his experiments is shown in Fig. ii6, taken from his paper. It is interesting to note that, like the curve of Gray for the oxygen consumption of echinoderm eggs, it shows no variations from its smooth course corresponding to the periods of cleavage. During the first 18 hours, the heat-production rate increases by four times, i.e. fairly parallel with the oxygen uptake rate. Membrane formation did not seem to have any effect on the heat-production, for eggs which had stood so long in sea water that they had lost the power of raising fertilisation membranes gave exactly similar results in the calorimeter. If the fertilisation membrane, however, was artificially produced in various ways, the heat production of the eggs might be raised considerably above the normal. The spermatozoa, which were also studied by Meyerhof, had an extremely small heat-production compared to that of the eggs; thus approximately 10 milliards of sperms gave off 4-6 cal. per hour when perfectly fresh, but after 3 hours in sea water this value had fallen to 3-1 cal. per hour.
Fig. 116.
The determination of the heat-production of the eggs was in itself very interesting, but Meyerhof went further, and compared it with the oxygen uptake of the same eggs, in order to obtain the calorific quotient, i.e.
Heat given off by i unit of material in gm. cal. per hour Oxygen taken in by i unit of material in mgm. per hour
This quotient, like the respiratory quotient, gives some sort of index of the type of metabolism going on inside the living cells under investigation. It was called by Pfliiger the "caloric coefficient of oxygen", and various workers had obtained the theoretical values for it, as follows :
Zuntz Rubner Pfliiger Average
For protein 314 3-0 3-3 -3-24 3-2
For fat 3-28 3-27 3-29 33
For carbohydrate ... 3-54 — 3"53-3'40 35
The differences are very small, and this probably accounts for the fact that less is heard of calorific than of respiratory quotients in the general literature. Nevertheless, in cases where the estimation of carbon dioxide output is difficult, such as eggs whose alkali reserve is unknown, the calorific quotient is very valuable. Meyerhof estimated it for normally developing eggs — leaving out of account eggs without membranes, etc. — as 2-75, 2-88, 2-675, 2-7, 2-675, 2-85, and 2-7; these were his seven quite satisfactory experiments. Roughly speaking, it wavered between 2-55 and 2-9, a value obviously much underneath the theoretical for any of the three main classes of energy source, although, as the lowest theoretical number was for protein, they lay nearer that than any of the others. These relationships are shown diagrammatically in Fig. 117. There was apparently no variation at all in the calorific quotient during development, as the following table shows:
Calorific quotient Unfertilised eggs ... ... 2-8, 2-525
I- to 2-cell stages ... ... 2-75, 2-775, 2-675
Morula stages ... ... 2-7,2-55
Swimming larvae ... ... 2-675
Blastulae 2-8
Some explanation was evidently required to deal with the marked lowness of all the figures. What made the situation still more perplexing was that these figures were uncorrected for the heat of solution of carbon dioxide in sea water and for other phenomena consequent upon the carbon dioxide output of the eggs. Meyerhof did not estimate the carbon dioxide directly, but calculated its effects from the few estimations which had already been done by Warburg, and, when this correction was made, the average calorific quotient sank to 2-6.
Meyerhof also found that, just as phenylurethane had been found by Warburg to inhibit cleavage while leaving respiration untouched, so it had no effect on the heat production, and, therefore, none on the calorific quotient. The calorific quotient of eggs in phenylurethane solutions was 2-65 to 2-75, a finding which conclusively showed that no chemical energy disappeared to form morphological structures, for, had that been the case, the calorific quotient must have been different when development ceased. Again, for parthenogenetic eggs with membranes raised by valerianic acid, the corrected
Fig. 117.
calorific quotient was 2-6. A more unexpected result was the calorific quotient of eggs in sea water to which ammonia had been added. Here, as Warburg showed, no cleavage goes on, but the respiratory rate is slightly raised. Meyerhof found that the calorific quotient of such eggs was 3-25 to 3-35, and this was the only case in which he got values at all resembling the theoretical ones. He was not able to devise a satisfactory explanation for this. Hypertonic sea water, which had a great effect on the heat-production rate, just as it had on the respiratory rate, made very little difference to the calorific quotient. In a sodium chloride solution of 4-3 per cent, it was 2-6, in one of 3-5 percent, it was 2-85, in one of 2-3 per cent, it was 2-9 (unfertilised) and 2-85 (fertilised). The calorific quotient of spermatozoa was found to be between 3-05 and 3-1, from which Meyerhof concluded that perhaps they were making use of protein as a source of energy.
Indeed, the question of the interpretation of the low calorific quotient was the main point of interest to Meyerhof. He examined the Strongylocentrotus eggs for glycogen and free glucose, and could find no trace of either^. Nor could he detect any nitrogenous breakdownproducts in the sea water surrounding the eggs (Nessler's reagent). But by the use of the Kumagawa-Suto method, he did succeed in revealing the presence of fatty substances in the unfertilised eggs, obtaining for an amount of egg-mass equivalent to 140 mgm. nitrogen, 1-905 gm. ash-free dry substance, a total ether extract of 0-323 gm., which contained 0-282 gm. of saponifiable fatty acids. This would correspond to 14-8 per cent, of fatty acid and 2-15 per cent, of lipoids and sterols (dry weight) . This material might then be used to supply the necessary energy. Meyerhof supposed that there were three possibilities: (i) That the oxygen was all being used for the oxidation of the fatty acids, but that at the same time certain strongly endothermic processes were going on which accounted for the missing 25 per cent, of the heat. In certain conditions, e.g. sea water containing ammonia, these endothermic processes would be considered to be abohshed, and the full amount of heat permitted to escape, giving a reasonable calorific quotient. (2) The oxygen consumed might not all be used for the combustion of the fat, but might partly be employed in synthetic processes without accompanying heat-production. We have already met with this idea in Rapkine's work. Lastly (3), the source of energy might not be exclusively fatty acids, but other substances, burning in exothermic manner, and sharing the total oxygen. In the two last-named cases the energy-content or calorific value of the eggs would decrease during development, even if some of the products of combustion were retained inside the cells, but, in the first alternative, this would not occur. Put in another way, the question would be, does the calorific value of the eggs decrease or increase, and, in either case, what relation does it bear to the wet and dry weights of the eggs. Evidently, the only way to ascertain what these relations were was to investigate the eggs during their development with the aid of a bomb calorimeter, and Meyerhof promised a study on these lines. But perhaps because of the great difficulties such a continuation of his work, which would have been extremely interesting, was never published.
- But see on this, Section 811.
To the three possible explanations of Meyerhof's low calorific quotients, however, a fourth might have been added, namely, the suggestion that, in spite of all his precautions, a consistent leakage of heat was going on in his apparatus. The great advances which have been made in recent years as regards the measurement of the heat-production of muscle tissue have shown how difficult it is to be sure of registering all the heat eliminated. Impressed with such considerations as these, Shearer in 1922 determined to re-investigate the matter. He criticised Meyerhof's technique on various grounds, e.g. use of too crowded cell-suspensions, insufficient aeration, reliance upon the Winkler method instead of on manometric methods. Shearer had already shown in the case of bacteria that the cytolysis of the cells was accompanied by a greatly increased oxygen intake and heat-production, so in this work he took special precautions against it.
His differential microcalorimeter was substantially the same as that elaborated by A. V. Hill for muscle, consisting of two vacuum flasks, a copper-constantan thermocouple, and a very sensitive galvanometer. Elaborate precautions were taken to prevent errors and to find the total amount of heat produced. Shearer felt that the most important result of Meyerhof's experiments was that whether he took the unfertilised egg, the fertilised egg, or the fertilised egg treated with phenylurethane, so that no cell formation was going on, although the egg was fully alive, he found that the value of the calorific quotient was always the same. Yet if any of the chemical energy liberated in the egg as the result of the increased oxygen consumption on fertilisation were utilised in producing the visible morphological structure of the embryo, the calorific quotient could not have been the same in all the instances.
Fig. 118 gives a graph showing one of Shearer's experiments. The calorimeter contained an amount of eggs corresponding to 58-4 mgm. nitrogen. In the ist hour after fertilisation the eggs liberated 2-9 gm. cal., in the 5th hour 10-5, and in the nth hour 22-8. All these figures, except the first one, are rather higher than the corresponding ones of Meyerhof, though it must be remembered that Shearer was working with Echinus miliaris and Meyerhof with Strongylocentrotus lividus. In another experiment where 146-2 mgm. Ggg nitrogen were present, 6-34 gm. cal. were given oflfin the ist hour, 28-0 gm. cal. in the 5th hour, and 74-4 in the nth hour. For the ist hour following fertilisation Shearer obtained a calorific quotient of 3-22, while for the unfertilised Q:gg his average figure was 3-07. He did not wish, however, to draw any conclusions from this diflference, for, in view of the large numbers of eggs which had to be used in the case of eggs before fertilisation, and the inevitable indi- o vidual differences between females in ripeness, physiological condition, etc., it was perhaps wise not to lay too great stress on the variation between the unfertilised and fertilised eggs. Nevertheless, the values obtained were much nearer the theoretical values than those of Meyerhof, so that it was not unlikely that these more accurate measurements had overcome the loss of heat which had led to his low calorific quotients. As Fig. 117 shows, Shearer's result worked out at about the level of protein combustion, although he himself laid no stress at all upon this fact; and certainly it did not agree very well with the respiratory quotient of 0-95 which he obtained simultaneously on the same material. What seemed to him of especial importance was that there was no marked difference between the calorific quotient of unfertilised and of fertilised eggs, and in this he confirmed Meyerhof's findings. He therefore concluded that only a negligible quantity of the energy liberated in the high metabolic intensity of the fertilised egg-cell was expended in bringing into being the visible morphological structure of the embryo. It was employed, he thought, in keeping the living substance itself intact as a physical system. Energy used for this purpose would presumably come under the heading of developmental work or "Entwicklungsarbeit", and this problem will be fully discussed in a succeeding section, but it may be said here that all the
Fig. 118.
Table 78.
evidence goes to show that it is very small proportionately to the general energy turnover of the embryo. At the same time, it remains a remarkable fact that the calorific quotient of the early stages of echinoderm development, at any rate, should be exactly the same no matter whether morphological differentiation is proceeding or not, i.e. before fertilisation and under phenyl-urethane, as against normal development after fertilisation. All these facts together with others which have been referred to above go to show the comparative independence of processes collaborating simultaneously in embryonic development to produce the finished organism. The developmental mechanisms do not function at the same rate or with the same rhythm, and the great problem of the future is the nature of their integration. At present we are only uncovering, as if by a kind of dissection process, the various contributory systems, such as those represented in Murray's diphasic schema for the chick. Shearer found that in i hour i million unfertilised eggs (corresponding approximately to 8 mgm. egg nitrogen) consumed 15-1 c.mm. of oxygen and gave off at the same time 0-067 gm. cal. at standard temperature and pressure. After fertilisation the same quantity of egg-substance consumed 86-4 c.mm. of oxygen and liberated 0-3976 gm. cal. heat together with an amount of carbon dioxide equivalent to a respiratory quotient of 0-92. These figures were afterwards gone over again by Rogers & Cole, who desired to introduce even more accurate methods, and to take readings at short intervals over considerable periods of time. Rogers & Cole only published one paper, most of which was taken up with problems of technique. In Shearer's experiments, the vacuum flasks were undergoing a gradual fall of temperature throughout the experiment, and the difference in rate of fall between the control flask and the flask containing the experimental material gave the heatproduction of the eggs. Rogers & Cole, however, used a different method, by which the fall was much slower, and therefore much longer experiments could
Fig. 119.
Fig.
be carried out. Fig. 119 shows one of their experiments. The total number of eggs used was 3-9 millions. The curve well demonstrates the absence of any variations due to cleavage. In Fig. 120 the average rate of heat-production in calories per hour per million eggs from all their experiments is given. It is probable that not much attention should be paid to the exact shape of the curve, but only to its general form. It is very interesting that the heatproduction rate should be for the first 3 hours after fertilisation a constant. This does not agree with the results of the other two workers, who found that the rate of heat-production rose during early development more or less parallel with the respiratory rate. The rate of heat-production of the unfertilised Arbacia e^g, according to Rogers & Cole, is o-o8 gm. cal. per hour, and during the 2-cell stage 0-52 gm. cal. per hour, figures which are distinctly higher than those found by Shearer and by Meyerhof.
Gm. cal. heat liberated per
I million eggs (8 mgm.
egg nitrogen) per hour
Before fertilisation (Meyerhof)
0-0514
„ (Shearer)
0-067
„ (Rogers & Cole) ...
008
After fertilisation (2-cells) (Meyerhof)
0-272
„ „ (Shearer) ...
0-40
„ „ (Rogers & Cole)
0-52
It is evident, however, that the percentage increase is just the same. Rogers & Cole drew no conclusions from these facts, but there is some likelihood that their figures are more accurate than those of the earlier workers, for, where slight losses of heat are the most probable cause of error, we ought perhaps to accept the highest figures as the best ones. It was obviously worth while to calculate calorific quotients for Arbacia again on the basis of the highest figures for heat-production, and this I did in 1927. Unfortunately Rogers & Cole never made any estimations of oxygen consumption on their eggs, and, although Loeb & Wasteneys stated in 19 10 that they had made measurements of respiratory rate on Arbacia eggs confirming Warburg's work, these seem never to have been published, so that the calculation could not be more than an interesting feeler. Using Shearer's figure for oxygen consumption (bearing in mind that it was obtained on Echinus, not Arbacia) and Rogers & Cole's figure for heat-production, the calorific quotient for the fertilised eggs (ist hour) worked out at 3-8, not 3-215, and for fertilised eggs (2nd hour) at 3-7, not 2-37. Similarly, for the unfertilised tgg, the calorific quotient worked out at 3-51, not 3-13 or 3*07. When these values are placed beside the older ones of Meyerhof and Shearer, as in Fig, 117, it can be seen that they overpass all the theoretical levels just as the earlier figures failed to reach them. There may be some significance in the fact that the calorific quotients thus calculated approach the carbohydrate level rather than any of the others, for, as will appear later, there is much evidence associating a combustion of carbohydrate solely or predominantly with the earliest stages of development.
4-5. Respiration of Annelid, Nematode, Rotifer, and Molluscan Embryos
Faure-Fremiet, in the course of his researches on the physiology of the egg of the polychaete worm, Sabellaria alveolata, carried out some determinations on the gaseous exchange, although, as far as I can find, his figures are not to be found in the literature in full. Using the Levy-Marboutin technique for estimation of dissolved oxygen in sea water, Faure-Fremiet found that the unfertilised eggs consumed almost as much oxygen as the fertilised ones. Thus 100 gm. of egg consumed in 100 minutes before fertilisation 42 mgm. of oxygen, and afterwards 47 mgm. of oxygen at 20°. At 19° the relative figures were 36 and 38, at 16° 15 and 16, at 11° 13 and 13, and at 0° 8 and 7. The feeble rise in respiratory rate (not more than 12 per cent.) appeared, therefore, to fade away as the temperature was lowered, a fact which led Faure-Fremiet to conclude that it was not of essentially the same character as the 800 per cent, rises invariably found in the case of the fertilised echinoderm egg. From these figures he calculated a temperature coefficient which was i-6 between o and 10° and 3-2 between 10 and 20°. As regards the rise in respiratory rate on fertilisation, it may, owing to loss in raising the cultures, etc., have been rather higher than the figures actually show. Faure-Fremiet also estimated the liberation of carbon dioxide from these eggs, using a modified form of the Osterhout-Haas method, i.e. titration of sea water in which the eggs have been respiring, to different end points with various indicators. Faure-Fremiet did not publish the figures he obtained by this method, and did not venture to calculate a respiratory quotient from them, but merely stated that "the relation between the values found is always near enough to unity to constitute a verification of the results observed, or at any rate of their order of magnitude". I do not quite understand what this means, but we may conclude that there is some evidence, at any rate, in favour of the respiratory quotient of Sabellaria eggs in the segmentation stages being about i-o. The resemblance between this figure and those obtained for segmenting echinoderm eggs will be evident.
The respiration of nematode eggs has also been studied by FaureFremiet, who employed Ascaris megalocephala as material, and the old-fashioned Bonnier-Mangin apparatus as technique. Fig. 121 shows the curves he obtained for oxygen intake and carbon dioxide output. These are for i gm. of dry weight, and so are true metabolic rate curves. They differ very much for those obtained on all other animals, for, instead of rising as development proceeds, as occurs both as regards respiratory rate and total amount respired in all other cases, they maintain a practically uniform level. Their absolute value approaches that of some fragmentary figures later given by Holthusen. One gram dry weight of vermiform embryos ready to hatch thus consume no more oxygen in 24 hours than one gram 20 of dry weight of newly fertilised eggs, a strange state of affairs, which may be related to the fact that, in later life, Ascaris differs from all the other examples in being eventually anaerobic. During development, Faure-Fremiet found that 50 c.c. of oxygen were absorbed and 43-8 c.c. of carbon dioxide given out per gram dry weight, and this led to some interesting calculations concerning the general metaboUsm, for which see later. The respiratory quotient for the whole period was 0-876, but, when it was calculated for each day during the 120 hours of development, the graph shown in Fig. 122 was obtained. During the earlier stages of development, the respiratory quotient fell from 0-82 at the 24th hour to reach 0-74 at the 72nd hour, after which it rose steadily, though more rapidly at first than later, to 0-92 at the end of development. It was therefore declining during the stages of segmentation and gastrulation, it reached its minimum at the time when the embryos were curved like a U, and it rose again during the main period of increase in length and the assumption of mobihty by the embryos. If, then, the Ascaris egg is burning its reserves completely to carbon dioxide and water, it could be stated roughly that a period of protein or mixed fat-protein combustion was succeeded by a period of carbohydrate or mixed protein-carbohydrate combustion.
Fig. 121,
Later work by Brown shows that a difference of temperature has no influence on the amount of oxygen taken up by nematode eggs during their development, but only affects the rate at which this process occurs^. Zavadovski has found that also in the case of the nematode tg^ {Ascaris) cleavage is stopped by lack of oxygen or by certain concentrations of potassium cyanide. He has brought forward evidence showing that this egg has two kinds of oxidation-processes, one group affected by potassium cyanide and the other not affected, and that cell-cleavage is associated with the former group. Reznicenko, like Zavadovski, has studied the effect of potassium cyanide (in large amounts) on the respiration of nematode eggs, but with paradoxical results.
Fig. 122.
Lite & Whitney have made some observations on the respiration of rotifer eggs. These do not normally hatch for many weeks after they have been laid, but if they are laid without a shell or with only a very thin one they develop fast, and hatch in a comparatively
short time. Lite & Whitney found that by vigorously aerating the water in which the eggs of Brachionus and Asplanchna were lying, they could make the thick-shelled ones develop as fast as the thin-shelled ones, and hatch as soon. They concluded, therefore, that the respiratory rate is much influenced by the amount of air available and that the developmental rate follows it.
- 1 This was confirmed by McCoy for the eggs of the hookworm, Ankylostoma caninum. These are exactly the same size as Ascaris eggs and take up the same amount of oxygen from fertiHsation to hatching, aUhough their speed of development under the same conditions is 21 times as fast. High oxygen tensions inhibit development of hookworm eggs.
Quantitative experiments were made by Buglia on the eggs oiAplysia limacina, the sea-hare, a gastropod mollusc. He employed Vernon's method for estimating the gases dissolved in sea water, which involves very elaborate apparatus, and gives a fair degree of accuracy, and he estimated the oxygen taken in and carbon dioxide given off. Fig. 123 a taken from his paper shows the curves which he got by plotting the cubic centimetres of oxygen taken in or carbon dioxide given out per kilogram of eggs per hour against the time from laying, all at three different temperatures. It will be seen that the carbon dioxide and the oxygen run closely parallel during the greater part of the time, and that there is a very marked rise in respiratory rate between the 30th and Goth hours. It is not easy to understand why at the lowest temperature this rise should be almost abolished, unless the time taken in development was then so long that it had not begun at the looth hour, by which time the other two curves had long attained a steady level. Nor does the upper curve (30°) agree with such a presentation of the data, for it would be expected to rise much more sharply than the curve at 20°, whereas, on the contrary, it rises more slowly. The respiratory quotients worked out as follows :
Table 79.
Respiratory quotient Hours State of embryo 10° 20° 30°
8 i-cell — o-8i —
12 44-cell — 0-85 —
13 Beginning of morula ... ... ... — 0-98 102
20 Morula ... ... ... ... ... — 0-72 —
25 First segmentations of nutritive blastomeres i-oo 0-90 —
27 Morulation of nutritive blastomeres ... — 0-90 o-88
30 Ectoderm nearly surrounds endoderm ... — i-o6 —
35 Ectoderm completely surrounds endoderm 1-25 0'94 0-92
50 Half-formed embryo ... ... ... — 1-19 —
60 Embryo quite formed ... ... ... i-oo i-i2 0-95
120 Embryo with many cilia ... ... ... 0-71 i-ii —
150 Embryo with rapid rotatory movement ... — 0-98 0-95
If the figures for oxygen uptake and carbon dioxide production during the 2 hours of an experiment were compared, it was found that in the early stages the eggs had a lower gaseous exchange during the 2nd hour than during the ist, but that in the later stages the reverse was the case, and the 2nd hour readings were greater than the ist. This was interpreted to mean a decreasing
60
cs 50
30
20
■bc 10
Temperature 30°C
Temperature 10° C
10 20 30 40 50 60 70 80 Time in hours Fig, 123 a (Buglia).
100 110 120 130
sensitivity with age to the effects of carbon dioxide in the ambient sea water. Returning to Fig. 123 a, the curve at 21° should be taken as normal, and from that it is clear that there are three periods in the change of respiratory rate with time in the case of Aplysia, {a) from o to 35 hours, {b) from 35 to 50 hours and [c) from 50 hours onwards. The point marking the transition of period {a) to period {b) is the complete surrounding of the endoderm by the ectoderm, while the point marking the close of the great rise is complete sketching out of the embryo with most of its parts. A study of Carazzi's monograph on the development of Aplysia does not reveal any more striking correlations. It is worth noting that Buglia's figures for metabolic rate must be accepted with the reservation that we do not yet know how the dry weight of the gastropod eggs varies during their development, nor how the total nitrogen behaves in relation to the total wet and dry weight. It is therefore not possible as yet to say how far Buglia's curves are comparable with those which have already been given for the metabolic rate of echinoderm eggs (i.e. related to so many mgm. of egg nitrogen). Nevertheless, it is interesting that the respiratory rate seems to increase as Aplysia develops, rising between two steady levels, just as that of echinoderms does. Whether these metabolic rates are at all comparable with those for the chick, for instance, is not sure, for in so many cases, e.g. amphibia and fishes, as well as this gastropod, it is \, either not possible or else very |f difficult to measure the actual g amount of protoplasmic sub- g^ stance at any given moment, 'i^ and the quantities of inert yolk | , must falsify the results a great |: deal. 1,
Meyerhof continued Buglia's work on Aplysia by making some determinations of its heat-production at different stages of development. He used the same apparatus as had been employed for his heat-production work on echinoderm eggs, and did oxygen estimations by the Winkler method. The calorific quotient worked out at 2-8 for the early and 2-9 for the late stages. Meyerhof confirmed Buglia's S-shaped curve for respiratory rate in these eggs (see Fig. 123^) and concluded from the calorific quotients that fat was being burned as a source of energy throughout development, pointing out the correspondence between this finding and the richness of the eggs in yolk compared with echinoderms.
Fig. 123 b (Meyerhof).
4-6. Respiration of Fish Embryos
A good deal of work has been done on the physiological and morphological side of the respiration of fish embryos, but only little on the physico-chemical side. As an example of the first type of work, the experiments of PoHmanti might be mentioned. He gave a good account of the first respiratory movements of the eggs of Scyllium canicula, and described their association with the development of the nervous system. Then a good deal is known about the probable respiratory function of structures in some of the rarer fishes; thus Ryder states that in the surf-perch, Ditrema laterale, the caudal fin has hypertrophied blood-vessels in utero which serve as respiratory portals for the embryo. This dermal vascularity disappears before birth^. Compare this with the similar structures in tropical land frog embryos described by Barbour. These questions will be further discussed in the sections on the placenta.
Quantitative observations began in 1896 with Bataillon, who measured the carbon dioxide evolved by the eggs of various kinds of teleosteans, such as the perch, the minnow {Phonixus laevis), the vaudoise {Luciscus jaculus), the rousse [Luciscus rutilus) and the gudgeon. He raised his minnow embryos in a current of moist air free from carbon dioxide, and not in water, and found that their development proceeded perfectly normally in such conditions. He concluded that there were two periods in development at which the carbon dioxide given off per hour was rather low, one at a stage just preceding the extension of the blastoderm over the yolk-sac, and one after the occlusion of the blastopore. In later experiments he actually placed the eggs in weak baryta, and found that they de ^ A remarkably interesting adaptation occurs in the case of the lung-fish, Lepidosiren paradoxa. The eggs develop in burrows where the water contains no measurable dissolved oxygen (Carter & Beadle) , but the male fish which guards the nest has long vascular filaments on its pelvic fins during the breeding-season, and these may secrete oxygen into the water around the eggs (Cunningham).
Fig. 124.
666 THE RESPIRATION AND [pt. iii
veloped normally, after which a titration at varying intervals was all that was necessary to give him his curve. In Fig. 1 24 are shown some curves constructed from the data which he obtained. They are neither smooth nor regular, but Bataillon, it must be remembered, was engaged in pioneer work. The continuous line is the curve for the minnow. The curves show, as he himself pointed out, (a) a rise during segmentation followed by {b) an accentuated fall during the extension of the blastoderm over the yolk-sac, after which the curve climbs again {c) to a peak at the end of the covering of the yolk by the blastoderm. Then there is a period {d) of lowering at the time of the occlusion of the yolk-plug, followed by {e) a slow rise till the beginning of active movement. It is difficult to know what emphasis to lay upon these results, for the description of the technique by which they were obtained is so short that no adequate idea can be had of it, and it presents, moreover, suspicious features.
It may be remarked in passing that the curves given by Bataillon are increment curves, and thus show up minor variations in the more or less regular curve relating time to oxygen uptake.
Scott & Kellicott in 19 16 made an extended study of the respiratory exchange of the embryos of another minnow, Fundulus heteroclitus. Unfortunately, this was never published, and all that we have is an abstract which gives nothing but the main points, and those almost too briefly to be of service. According to Scott & Kellicott, in the early cleavage stages 1000 eggs use o-i c.c. of oxygen per hour. The appearance of the circulation of the blood produces a marked rise, after which another steady level supervenes, so that at hatching they are using 0-7 c.c. of oxygen per hour, having consumed throughout the entire period from fertilisation to hatching about 80 c.c. Six days after hatching they use 1-75 c.c. per hour. From fertilisation to hatching 38 per cent, of the egg-weight is lost, presumably by combustion. One thousand eggs in early cleavage stages were found by Scott & Kellicott to consist of 0-12 gm. of protoplasm and 2-65 gm. of yolk, but 6 days after hatching they weighed i-8 gm.; 0-12 gm. of protoplasm used o- 1 c.c. of oxygen per hour, so that the metabolic rate for the early cleavage stages was 8-34 c.c. oxygen per hour per 100 gm., and from the figures given for 6 days after hatching it was 9-74 c.c.
Hyman also worked on Fundulus eggs with the Winkler method.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
667
Fig. 125 shows the undulatory curve which she obtained in her experiments, with peaks at 2, 4 and 10 days after fertiHsation. The values, it will be noted, are of the same order as Scott & Kellicott's. The segmentation stages were all passed through during the first upward rise of the curve, which reaches its peak at the time when the morula stages (though the term is inappropriate for a teleostean have all been passed through, and the shape of the embryo
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668 THE RESPIRATION AND [pt. hi
be feared that the number of points secured by Hyman is insufficient to establish so sharply inflected a curve. It gives us little indication concerning the metabolic rate for we do not know the rate at which the non-respiring yolk is disappearing, but Hyman did not hesitate to conclude that " the respiration is probably highest per unit weight of protoplasm early on the second day of development since from that time on the amount of protoplasm increases greatly but the oxygen consumption does not increase in like proportion, in fact, a considerable part of the oxygen consumption after the third day is due to the activity of the heart. As the embryo is continually increasing in size after this time while the oxygen consumption per hour shows little increase relatively, we may reasonably conclude that the oxygen consumption per unit weight of the embryo is actually decreasing". Hyman also made estimations of the carbon dioxide production, observing the time required for 40 eggs to take a given amount of sea water from pH 8-2 to 7-6, but these she did not publish, simply stating that "the study of the CO2 production yielded similar results. It increased per unit time up to the early part of the second day of development after which it fell, rising again in later periods".
A comparison between the results of Hyman on the oxygen consumption of Fundulus and Bataillon on the carbon dioxide production of Phonixus is of interest. Bataillon's work was apparently unknown to Hyman, but it can hardly be mere coincidence that the general tenor of the curves should be the same. The first peak in Hyman's curve is on the 2nd day, so is the first peak on Bataillon's curve, the great trough on Bataillon's curve presumably corresponds with the low values obtained by Hyman on the 3rd day, and all the later values show a suggestive though not close correspondence. However, from the description given in each case, there is some doubt as to whether the underlying processes are going on synchronously, and doubtless the time of development in the two minnows differs. It may also be significant that Tangl & Farkas's few points for the carbon dioxide output of the trout egg show a slackening of the rise just at the time of Hyman's first trough.
The respiration of the plaice egg {Pleuronectes platessa) has been investigated by Dakin & Dakin and by Burfield. Dakin & Dakin made careful analysis of the eggs at two points in their development, at a point between the 8- and i6-cell stages and at 14 days after
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 669
wards. During this period 2000 eggs consumed between 82-086 and 98-00 mgm. of oxygen, and when this amount was compared with the amount of solid matter used up during incubation, the results were very close, for the eggs lost 78-3 mgm. of protein and the oxygen used accounted for between 65-67 and 77-8 mgm. Further consideration of the work of the Dakins will, however, be deferred to the sections on General Metabolism, Energy Sources, etc. Burfield investigated the respiration of plaice eggs with a view to finding out whether the gaseous exchange fell off during a single experiment in the closed chamber, and, if so, why. It is extremely tantalising that he apparently made no record of the age or state of development of his eggs, classing them simply as "young"; his figures which might, therefore, have been useful, as showing differences in respiratory exchange with age, have no value for our main purpose. The fall in the rate of oxygen consumption of aquatic organisms might be due, he argued, to a combination of four factors: (a) handling of the animal at the beginning, (b) absorption of the available oxygen, (c) accumulation of carbon dioxide or other excreta and {d) feeding having gone on immediately before the experiment. Factors {a) and (d) do not operate in the case of eggs, and factor {b) was avoided by using a sufficient volume of sea water. Accordingly Burfield found that the third possibiUty was very important, and was able to depress considerably the rate of oxygen consumption of the developing plaice eggs by adding small amounts of carbon dioxide to the water. They were far more sensitive to this than to reduced partial pressures of oxygen. Urea had no effect. If the eggs were frequently moved so as to prevent accumulation of carbon dioxide in their immediate vicinity no fall in oxygen consumption was detectable, and the amount absorbed during the ist hour would be a fair average of the values for the succeeding hours. Whitley had already noted that the amount of variation from the normal pH which plaice eggs will tolerate is very small indeed, and that a disturbance of the equilibrium towards the acid side is much more fatal than a disturbance towards the alkaline side. These facts fit in together well, but Whitley's work was not altogether confirmed by Hopkins, and the latter actually found that eggs of the trout and perch would not develop properly in the absence of very small amounts of free carbon dioxide. Burfield also occupied himself with the respiratory quotient of the eggs, measuring the carbon dioxide
670
THE RESPIRATION AND
[PT, III
produced by an indicator method, and the oxygen taken in by the Winkler method. In one case the respiratory quotient was 0-78 and in another case 0-72. We are not informed what the age of the embryos was in these experiments, but the second figure was obtained from a batch which was "younger" than the batch which gave the first value. These respiratory quotients are in fair agreement with the fact now definitely known, that there is a very large expenditure of protein in the egg of the plaice to furnish energy during development.
Kawajiri has studied the embryonic respiration of the Japanese landlocked salmon, Oncorhyncus masou. Apparently the oxygen consumption per fish per hour rises steadily until hatching, after which there is a rapid increase, followed by a continued slow rise at much the same slope as before hatching, until the yolk has disappeared, after which time other factors come into play.
The best work on the respiration of the fish embryo is that of Gray , who in 1926 measured the respiration of the brown trout, Salmofario. His experiments did not begin till the 46th day from fertilisation, about which time the fish escapes from the egg-envelopes and swims freely, existing on the stores in its yolk-sac. The graph given in Fig. 1 26 shows the figures obtained by Gray for metabolic rate. Assuming that the substances combusted were partly fat and partly protein. Gray calculated that the amount of oxygen consumed over the period in question was exactly equivalent to the amount of dry material disappearing from the system. Hayes subsequently studied the respiration of the eggs of Salmo salar. It fell from 0-075 g'^- oxygen per 1000 eggs per hour on the 40th day to 0-045 on the 65th day.
In 1928, Boyd, working with the egg of the minnow, Fundulus heteroclitus, and using three methods on the same material (Winkler titration, Fenn's modification of Barcroft's manometer, and the Haldane gas analysis apparatus), found that there was a marked rise in oxygen consumption in the egg after fertilisation. This was an
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SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
671
important advance, for although it had always been considered that the Warburg-Meyerhof-Shearer experiments on echinoderm eggs had the stamp of universality about them, there was no evidence that the rise in oxygen consumption at fertilisation held for any other phylum. Plotting the oxygen consumed by her minnow eggs per unit weight in each lo-minute period, against the time, she found that it rose to a peak 60 minutes after fertilisation, at which time it was 1 7 times the unfertilised egg value. Then, falling past the time of first cleavage (120 minutes) it reached the unfertilised egg value at 210 minutes. Boyd did not take any readings after the 310th
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4-7. Respiration of Amphibian Embryos
Bataillon was also the first to make quantitative experiments on the respiratory exchange of developing amphibian eggs. His papers already quoted are interesting, in that they form one of the most detailed attempts at correlation between morphological and biochemical data which have ever been made. In this they suffer from two disadvantages, firstly that Bataillon's methods would not be regarded now as accurate although Hyman's work is to some extent confirmatory, and secondly that he made one or two doubtful theoretical
672 THE RESPIRATION AND [pt. iii
assumptions, the nature of which will presently become clear. In order to understand his point of view, attention should again be directed to Fig. 124, in which his data are presented for the carbon dioxide given off by the embryos of the minnow and the frog during their development. Both show the preliminary rise on segmentation, but the frog curve descends sooner, and then rises steadily to reach a peak about 60 hours after fertilisation, at the same time as the minnow. From that point onwards, the amphibian and the teleost run closely together. The development of the frog, as Bataillon pointed out, is, in fact, more rapid in the early stages. The occlusion of the yolk-plug occurs earlier relatively in the frog than in the minnow, yet the subsequent work of embryonic organisation takes a longer time in the former than in the latter. In the frog, between the initial segmentations and the period of spread of the ectoderm over the yolk there is, as can be seen, a short trough, after which the respiratory activity mounts steadily till the time comes for the closure of the yolk-plug. In the teleost, on the contrary, the period of extension of the blastoderm is short, and is preceded by a well-marked stasis, as if the development was meeting a persistent and not easily conquered obstacle. This once overcome, the extension of the blastoderm over the yolk goes on rapidly, accompanied by the rapid upstroke of the respiration curve. The embryo of the minnow having arrived at the 1 8th hour is constantly enriching itself with new cells, especially on its under surface. The periblastic elements are exercising a kind of sorting action on the yolk, by which it furnishes an abundance of chromatin material for future cell-divisions. But towards the 30th hour the spider-Uke forms of cells characteristic of the earlier stage have disappeared, and there is a very sharp boundary-line between the yolk and the periblast, all the cells of which appear to be at rest, very few mitotic figures being visible. In the embryo itself exactly the same state of affairs has come about ; the cell-divisions are very rare even on the under surface, where they are mostly to be found at the edges. There is, in fact, a period of temporary cessation of mitotic activity, and of slowing down of developmental rate. Bataillon thought that the reason for this was the heaping up of the layers of cells, as many as fifteen being demonstrable in certain regions, so that the inner ones were cut off from the air, and the outer ones from the nutritive materials of the yolk. In such circumstances the metazoal embryo might be considered to have gone as far as it
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 673
could go in the absence of a circulation. Bataillon pointed out that this temporary cessation of mitotic activity corresponded exactly with the trough in carbon dioxide production shown for the teleost in Fig. 124, between the 40th and 50th hours. The mitotic divisions having localised themselves round the edge of the embryonic area, the central part becomes dislocated, or rather raised up, and the deep cells, many of which show amoeboid processes, migrate to the periphery. Between the raised up germinal area and the periblastic layer a fluid appears, thus making a very irregular segmentation cavity, in which the deep-lying cells, finding better conditions, begin to proHferate and form the primitive endoderm. These cellular displacements and migrations, these mechanical difficulties, due to the beginning of extension of surface, said Bataillon, are probably the obstacles which give to that particular stage in teleostean development its peculiar characteristics, among the most striking of which is the trough in the carbon dioxide production. Then, after this stage, the extension of the blastoderm begun by the proliferation of the cells at the edges of the germinal area goes on continuously until the yolk is completely covered. In the case of the amphibian embryo, none of these difficulties arise, for the extension of the ectodermal elements at the animal pole over the yolk-laden cells of the vegetal pole is a relatively simple process.
In commenting on Bataillon's investigations, the resemblance between his curve and that of Hyman must be carefully considered, for, although the time correspondence is sufficiently close to warrant the belief in a real agreement, the descriptions given by the two writers are slightly at variance. Phonixus, according to Bataillon, accomplishes its surrounding of the yolk by the blastoderm after the great trough, but Fundulus, according to Hyman, does it partly before. Again, the curve of Hyman, though of the same shape as Bataillon's, is more lengthened out along the time axis, so that the peaks and troughs do not exactly correspond. Balfour, moreover, does not describe the static condition on which Bataillon lays such stress. It is therefore difficult to appraise Bataillon's results. As regards the theoretical side of his work, it might perhaps be observed that his simple direct correlation between amount of carbon dioxide put out per hour and number of mitoses going on is not altogether satisfactory. We have no evidence that there is a general connection between these two phenomena. Then one might ask why cell-migration should not be
674
THE RESPIRATION AND
[PT. Ill
just as much associated with high respiratory rate as mitotic activity. The question is in a confused and unsatisfactory state, and further researches with more accurate methods are greatly to be desired.
Another pioneer worker on the respiration of the amphibian embryo was Godlevski, who published his work in 1900 in connection with the susceptibility of frog's eggs to oxygen want. His technique was rather better than Bataillon's. The data he obtained are shown in Fig. 128, and consist of two smoothly ascending lines composed of rather scattered points.
Nothing further was done on amphibian embryo respiration till 1915, when Bialascewicz & Bledovski attacked the question, using the Winterstein micro-respirometer, a great advance on the technique of the earlier workers. The eggs of Rana temporaria 2.0 were used. Bialascewicz & Bledovski found that, 1.5 during the first few hours after laying, unfertilised 1.0 eggs hberated a "neutral gas " which, as far as could 0-5 be ascertained, was a mixture of oxygen and nitrogen. This doubtless arose from the difference in environment as regards gases between the ovary in the female body and the water outside. One thousand eggs gave rise in this way to a positive pressure of I2'i mm. 16 minutes after laying, and 1-15 mm. 47 minutes after laying, but thenceforward the pressure was constantly a slightly negative quantity. In order to avoid technical errors which arose naturally from this fact, Bialascewicz & Bledovski compensated the gaseous exchange of one lot of eggs in one vessel by having an identical quantity of eggs from the same female in the other vessel. Another phenomenon seen in the unfertilised eggs was a notable production of carbon dioxide immediately upon laying. Eggs taken from the lower part of the oviduct and brought straight into the micro-respirometer eliminated large amounts of carbon dioxide, presumably because the tissues of the frog with which the eggs had previously been in gaseous equilibrium were saturated with carbon dioxide. This carbon
Days-^
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144
168
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Fig. 128.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 675
dioxide output declined rapidly after the eggs had been taken from the oviduct or had been laid, and by 100 minutes had reached a steady low level. One thousand eggs 50 minutes after laying produced upwards of 20 c.mm. of carbon dioxide per minute, but after the looth minute was reached, their steady level was about i c.mm. carbon dioxide per minute. McClendon's centrifugation experiments already referred to showed that 16 per cent, of the frog's egg was "clear and protoplasmic", 6 per cent, was fatty or oily and 78 per cent, was yolk. As an average egg of Rana temporaria weighs 3-43 mgm. (Bialascewicz & Bledovski), the amount of protoplasm in it would weigh 0-55 mgm., or in 1000 eggs 550 mgm., and this amount at I c.mm. carbon dioxide per minute (wet weight) would be o- 1 82 c.mm. carbon dioxide per 100 mgm. protoplasm which compares interestingly with Rapkine's 0-225 c.mm. carbon dioxide per 100 mgm. in the sea-urchin's egg. But this calculation is not very significant, owing to the manifold uncertainties involved in taking data from many authors on different material in different conditions. It does perhaps show a similarity between the metabolic rate of the unfertilised egg of echinoderm and amphibian. The conclusion from this part of Bialascewicz & Bledovski's work was that during life in compact masses in the oviduct the eggs accumulate large quantities of carbon dioxide, which has to be eliminated when the eggs are brought into an atmosphere containing a much-diminished concentration of carbon dioxide. If this process were to go too far fertilisation would become impossible, as Bialascewicz & Bledovski showed by subjecting the unfertilised eggs to atmospheres of pure carbon dioxide for as little as 2 hours, and these findings do indeed go a long way towards explaining the phenomenon of prematuration The average results for oxygen uptake and carbon dioxide production of unfertilised and fertilised eggs worked out as follows :
Carbon dioxide Oxygen used
liberated by
up by 100
100 eggs per
eggs per hour
Respiratory
hour (c.mm.)
(c.mm.)
quotient
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6-57
12-86
0-511
6-07
8-90
0-682
Average ... 6-31
10-58
0-607
[Gastrulation 24-4 (single experiment of
Saunders)]
676 THE RESPIRATION AND [pt. iii
It is very interesting to note that the respiration shows a distinct increase after fertilisation, the extreme instance being a rise of two and a half times in the case of carbon dioxide and of nearly three times in the case of oxygen.
Turning now to the respiratory quotients, it is evident that there is not much difference before and after fertiUsation, a fact which is interesting in view of the constancy of the calorific quotient in echinoderm eggs. Bialascewicz & Bledovski admitted the difference between o-6o and 0-72 the theoretical fat respiratory quotient, but claimed quite justifiably that in many other cases where vigorous catabolism of fatty acids is known to be going on, the respiratory quotient is often below 0-7. At the same time, they saw in the lowness of it in this case evidence for a simultaneous activity of hydrolytic processes, both before and after fertilisation, though they did not give any more detailed indication of the part they supposed these to be playing in the metabolism of the embryo,
Bialascewicz & Bledovski went on to measure various entities in the egg and the early stages of development. Weighing, they found, presented great difficulties, so they measured the diameter microscopically, calculated its surface and its volume which, when multiplied by the specific gravity (1-102), gave the mass. From these investigations the following figures resulted :
Oxygen taken up by unfertilised eggs
Per 1 000 eggs Per i sq. Per 1000 gm. (c.mm.) metre surface egg-weight (c.c.) (c.c.)
Averages of 8 experiments ... ... ... 74-5 7-1 21-6
Percentage scattering of individual observations 330 19-2 i8-o
From these measurements it appeared that the smallest deviation from the average value resulted when the oxygen taken up was referred to the actual weight of the eggs, though evidently the surface is nearly as good a measure. This shows that the oxygen uptake depends not so much on the quantity of individuals measured as on their surface, or even more, on their weight. The cubic centimetres of oxygen absorbed per kilo of unfertilised egg-weight may be compared in an interesting way with the cubic centimetres of oxygen absorbed per kilo of adult frog (Bohr) ; the figures are respectively 21-6 and 261-8, so that the metaboHc rate is evidently far higher in the adult frog than in the unfertilised frog's egg.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
677
Bialascewicz & Bledovski next studied the intake of oxygen during the early stages of development, and their results are shown in Fig. 1 29. The curve rises smoothly during segmentation and gastrulation, etc., but about the 150th hour attains a plateau. This plateau is maintained as long as the tadpoles are kept without food, but when they begin to eat the respiration rises in amount again. Bialascewicz & Bledovski rightly laid special stress on their confirmation of Warburg's results, in that the respiration does not rise pari passu with the increase in general mass of nuclear substance. In absolute amounts the values for oxygen intake of Bialascewicz & Bledovski are almost double those of Godlevski, but all these investigators agree in the general upward trend of the curve and the absence from it of the undulations found by Bataillon. They found that the rising part of the curve, i.e. the plateau, could be expressed by an equation for a parabola, before i.e. x^ kt^ + a, where x is the amount of oxygen taken up by a given number of eggs during time t, a the value for unfertilised eggs, and k a constant. This rise then proceeds in a manner directly proportional to the square of the developmental time. Bialascewicz & Bledovski applied the same equation to the figures obtained by Meyerhof for the respiration of the sea-urchin and by Bohr & Hasselbalch for the chick. In the case of the frog, the constant was 0-0139, and the agreement between observed and calculated values was very fair, as follows :
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678 THE RESPIRATION AND [pt. m
The matter was then taken up by Parnas & Krasinska, who pubHshed their resuks in 1921. On many points their work led them to other conclusions than those of Bialascewicz & Bledovski, for they could not find respiratory quotients corresponding to the combustion of fat, and they did not obtain perfectly regular curves for oxygen consumption. They used as their principal method the Barcroft manometer technique, and their experiments seem to have been better carried out than those of any of their predecessors. The/ worked on the eggs of three species of amphibia, Rana temporaria, Bufo variabilis, and Rana esculenta. The experiments included more elaborate controls than had before been made, e.g. it was ascertained that the presence or absence of the gelatinous coverings made no difference to the respiration of the embryos in any stage, and that the development was normal in all the experiments, irrespective of the partial pressure of oxygen within wide limits. After 70 hours at 15°, the embryos which had been in a pure oxygen atmosphere were slightly more advanced than those which had been in the air, but at 11° this difference was hardly noticeable, although the diffusion of the gases would hardly be affected at all by such a change of temperature. Experiments were carried through at the higher temperature when it was desired to follow a long period, but at the lower temperature when the details of the early stages were under examination.
Parnas & Krasinska found that the oxygen consumption of Bufo vulgaris eggs at 14° in air before fertilisation was o-og c.mm. per Ggg per hour, but after fertilisation 0-34 c.mm. per Qgg per hour, a rise of about four times, rather more than had been found by Bialascewicz & Bledovski on the frog. In pure oxygen the rise occurred just the same, and to the same extent, though the absolute figures were higher. Parnas & Krasinska regarded the unfertilised egg as a dying cell, in view of the fact that, at a definite time after laying, it loses its capacity for being fertilised, and they suggested that the asphyxial conditions which had been shown by the earlier workers to hold in the oviduct were important as conserving the eggs in a state of suspension, it being impossible for any great amount of metabolism to go on in them before laying.
Typical graphs of Parnas & Krasinska's results are given in Figs. 130, 131 and 132. The first of these shows the oxygen uptake of the eggs of Rana temporaria for the first 1 00 hours after fertilisation ;
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
679
this curve is not an increment curve, but shows the total amounts consumed up to each time point, in cubic milHmetres of oxygen.
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The second figure shows the same thing for the eggs oi Rana temporaria for the first 70 hours after fertiHsation, and in the third figure is shown an increment curve, i.e. the amounts of oxygen consumed per hour by 1000 eggs. As is evident, the rise in respiratory rate
68o
THE RESPIRATION AND
[PT. Ill
is not regular, but has certain definite points of transition, and tends to approximate rather to a series of straight Hues than to a segment of a curve. The findings of Parnas & Krasinska, then, were in a sense a return to the original interrupted curves of Bataillon. The respiration of the amphibian embryo, said Parnas & Krasinska, during the segmentation, morula, and blastula stages, is proportional to the time passed and uniformly rising. The increase in the number of cells is certainly accompanied by an increase in the respiration. But at
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Hours Fig. 131, ( Upper line = in air; lower line = in oxygen.)
the time of gastrulation, there is a marked rise in the gaseous exchange (see Fig. 132), and a further powerful acceleration of it is seen at the time of the formation of the medullary plate, i.e. after the embryo has formed its longitudinal axis. "The first cleavage processes", said Parnas & Krasinska, "and the dividing of the embryo into potentially different cells proceeds with a uniform amount of respiration, but the differentiation of the germ-layers first of all, and then, the formation of structurally and chemically different cells, are accompanied by enhanced metabolic intensity and therefore by an increased respiration. After the neurula has been formed there is little
i
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
68 1
or no change in the respiration until the external gills appear, at which stage there is a third increase of oxygen-uptake. The succeeding period is again marked by a uniformity of rise." The three critical points in amphibian development, then, according to Parnas & Krasinska, are (i) gastrulation, (ii) formation of medullary plate, neural groove, etc., and (iii) appearance of external gills. These relations can best be seen on the increment curve in Fig. 132, where the breaks at gastrulation and the formation of the neural groove can be seen well.
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Parnas & Krasinska considered the question of whether their sudden increases in respiration could be accounted for by changes in the surface-volume relation or other causes of non-metabolic origin. At the neurula stage, for instance, when the spherical form of the embryo is abandoned, a greater oxygen consumption might be supposed to be due to the consequent increase of surface. But they concluded that the operation of such factors did not account for their results, since gastrulae respired no more in pure oxygen than in air.
Parnas & Krasinska suggested as the cause for the low respiratory quotients found by Bialascewicz & Bledovski processes in which oxygen was combined in the materials of the cells under construction — the same explanation as Meyerhof had already advanced. If they were indeed accurately measuring the carbon dioxide output of the
682 THE RESPIRATION AND [pt. m
embryos, and such factors as alkali reserve, etc., were not exerting too great an effect, then the Meyerhof theory is certainly more satisfactory than the one suggested by Bialascewicz & Bledovski. But it remains to be shown that absolutely all the carbon dioxide produced was being successfully measured. That much fat is burnt during amphibian development was definitely denied by Parnas & Krasinska on the basis of their actual estimations of fat and protein during the embryonic period, but this aspect of their work must be reserved for discussion later. According to their view, the segmentation period involves no more than a distribution among cells of protoplasmic constituents already in existence in the egg, and it is not until the separation of the embryo into the three germ-layers that intensive chemical work begins to take place. It is this that leads to rise in respiration.
Frog's eggs, it seems, can be anaesthetised, and Irwin has studied the consequent variations in their carbon dioxide output.
4'8. Heat-production of Amphibian Embryos
Only one examination of the heat-production of amphibian eggs exists in the literature, namely, that of Gayda, who described a differential microcalorimeter of great accuracy which could be used for small objects. With this instrument he measured the heatproduction of the eggs of the toad, Bufo vulgaris, throughout their development from fertilisation to hatching. Ruffini had previously ascertained that small amounts of water in which the eggs of Bufo vulgaris were developing had usually a temperature of from 0-5° to 0-6° at 10° and from i-o° to 1-5° at 20° higher than control quantities. Gayda confirmed many other workers on other material by finding that the heat-production did not rise pari passu with the increase in nuclear material or in number of blastomeres. Fig. 133, taken from Gayda's paper, shows the curve which he constructed from all his average results, where gram calories of heat evolved per gram of embryo and larva per hour is plotted against time, i.e. days from fertilisation. The arrow pointing downwards marks the point of hatching, and towards the 120th day metamorphosis begins. The shape of the curve is very regular and striking. Rising smoothly from the moment of fertilisation, and so continuing unaffected by the hatching process, it reaches a blunt peak at about the
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
683
20th day, after which it slowly declines, never falling, however, below about a third of its maximum value. This curve is, of course, a true measure of metabolic rate, and obviously fits in well with the work of Parnas & Krasinska, and of Bialascewicz & Bledovski. We here see for the first time a possible reconciliation between the apparently conflicting behaviour of the metabolic rate in various types of embryo. As far as can be ascertained at present, the metabolic rate in the echinoderm and the amphibian embryo rises during its ontogenesis, yet there is ample evidence, as we shall see later, that the metaboHc rate of the avian embryo consistently falls, at any rate from the 4th or 5th day of incubation onwards.
1-0 ^0-9
S3 0-7
-0.6
h 0-4 ^0-3 I 0-2
o
c
^ ^
^^
r
«,
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/
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-^
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if
^
10 20 30 40 50 60 70 80 90 100 110 120 130 Days after fertilization
Fig. 133.
Possibly the curve for the heat-production of the toad embryo gives the clue in suggesting that in all embryos there is a point at which the metabolic rate is higher than at any other time. The investigations of the extremely early stages in the echinoderm egg which we have been discussing have on this view revealed only the upward stretches of this curve, while the work on the chick embryo, which it must be remembered has completed its gastrulation before the egg is laid at all, has shown us the descending part of the curve. In the mammal, moreover, the curve for heat-production per gram per hour follows a peaked course ; for instance, the work of Wood and his collaborators has revealed very accurately the time at which this takes place in the pig. Two points must not be lost sight of in this discussion, firstly, that, for questions such as these, no hard-and-fast line can be drawn between embryonic or foetal growth and postnatal growth; the act of birth or hatching may be relatively
684 THE RESPIRATION AND [pt. iii
unimportant, and the fundamental waves of metabolism, growth, and differentiation, may pass through the individual organism without paying much attention to it. Secondly, although it is our duty to regard as many kinds of embryo as possible as special cases only of a few profound and general rules, we cannot escape the fact that their origin is very different, and it may not at present be possible to speak of holoblastic and meroblastic eggs, for instance, on the same basis. Thus the anomalous case of Ascaris eggs, in which there is no rise of metabolic rate during segmentation, etc., must be remembered. I shall return to these questions at the end of this section, when the data for the bird and the mammal have been presented.
Gayda's curve shows that shortly after fertilisation i gm. of toad embryo liberates 0-037 §"^- cal. per hour, while at hatching it liberates about 0-30 gm. cal. per hour, and at its maximum (20th day after fertilisation) it liberates as much as 0-97 gm. cal. per hour. Gayda did not himself calculate any calorific quotients, for he did not himself make any estimations of oxygen uptake, nor had at that time Parnas & Krasinska's work on amphibian embryos been published. Unfortunately, although they worked with Bufo vulgaris, none of their published protocols refer to that organism, but all to Rana temporaria and Rana esculenta. It is therefore not possible to put the figures together. The total amount of heat given out by I gm. of embryo (wet weight) throughout the embryonic period (fertilisation to hatching) was 30-276 gm. cal., and the corresponding quantity for i embryo was 0-1207. These values are the " Entwicklungsarbeit " of Tangl, relative and simple respectively (see p. 950). Gayda pointed out that the figure of about 30 gm. cal. was not nearly so great as the corresponding values for the chick found by Tangl & von Mituch, and for the silkworm by Tangl & Farkas, of about 900 gm. cal., but, on the other hand, it was about equal to Faure-Fremiet's figure for Sabellaria alveolata. These questions of energetics will be dealt with fully in the section on that subject. Between hatching and the end of metamorphosis 1668 gm. cal. were given out per gram wet weight, and 67 gm. cal. per larva. This covered 123 days. Thus the average heat output per gram wet weight per day before hatching was 3-75 gm. cal., and the average afterwards was 13-58, a striking result of the peak which occurs after hatching.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
685
Gayda, attempting to explain the slow fall in heat-production and metabolic rate after the 20th day, discussed the relations between surface and volume. As the larva grows in volume so its surface must proportionately diminish. What is so striking about the peaked curve for metabolic rate is that in the very earliest stages of development, while segmentation and gastrulation are proceeding, the heatproduction per gram per hour is increasing in spite of the fact that every moment the surface is diminishing in proportion to the volume and the weight. After the main inflection in the curve a simple surface heat-radiated relation is conceivable, but not before it. It is probable, of course, that the dermal surface is not the active surface, or rather not completely coterminous with it. An immense field of study exists in the determination of the surfaces in the growing embryo and the identification of the active one. Gayda found that, after the 20th day, if the gram calories produced were related to the surface of the embryo (calculated by the VW^ formula) the result was almost a constant, though at first there was some divergence. Thus about the 25th day 100 sq. mm. radiated 0-211 gm. cal. per hour, but on the 97th day 0-171 gm. cal., and on the 131st day 0-169 gm. cal. In fact, the gram calories liberated per square milHmetre per hour form a curve which declines from the 25th day; this is represented in Fig. 134. If it is compared with Fig. 166 a, in which the calories radiated from I sq. metre per hour are plotted against the age in the case of the pig and the human being, the resemblance is very striking. I shall return to this point. Gayda himself did not see anything important in this peak, however, and considered that it was probably due, on the one hand, to the change in shape of the embryo from spherical to axial, and, on the other hand, to the first swimming movements of the embryo about the time of hatching, believing that, if a constant could be introduced into the calculations to allow for such changes, the peak would entirely disappear. This may or may not be the case, and it is
Days from fertilisation Fig. 134.
686
THE RESPIRATION AND
[PT. Ill
also necessary to remember that the unabsorbed yolk-mass will in the early stages be included in the weight estimations, though it cannot be counted as thermogenetic tissue. It is easy to understand the decline in metabolic rate with advancing age, for the surface, i.e. the means of exit from and entrance to the body, does not grow as fast as the weight, but it is difficult to understand, on the view held by some physiologists that thermolysis is the cause of thermogenesis, how the embryo can have an increasing metabolic rate in the early stages — as it assuredly does — when every moment the surface/volume ratio is diminishing. The factors controlling the production of heat must be sought somewhere within the body rather than at the surface.
Gayda also discussed the interesting relations that exist between the heat-production and the time required to double the weight. Curves for these values are shown in Fig. 135. The S-shaped t nature of the curve is striking, ^ but perhaps the lowering at the f older stages is brought about by I metamorphosis, and so lies out | of the strict part of this discus- .| sion. During the greater part of ^ the larval period, both before ^ and after feeding has com- 5 menced, however, the parallel- J ism between the two curves is close. Obviously, the less heat that is evolved during the doubling of the weight, the more efficient will be the embryo or larva, and the more economically the turnover will be progressing. Fig. 1 35 shows that the least heat is evolved in the earliest stages, i.e. shortly after hatching, so it must be concluded that the greatest efficiency exists then. The most inefficient point would appear to be at the weight of 40 mgm. Whether it is significant that just at this point feeding begins is not clear. These relations are the direct opposite of what has been found to hold in the case of the chick, which at the 3rd day of incubation appears to be very inefficient, but which attains a maximum efficiency a few days before hatching. A full treatment of this point is given in Section 7-5.
Gayda also calculated the temperature coefficient of the heat production, and found that it worked out at an average of about 2- 10,
10 20 30 40 50
Wfc. of embryo or larva in mgms.
Fig. 135
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 687
but his data at different temperatures are hardly sufficient to allow of the calculation of a temperature characteristic. The total quantity of heat lost, however, remained quite constant during periods of doubling of weight at all temperatures at which normal development proceeded; a finding which was later to be confirmed very fully on the frog by Barthelemy & Bonnet using bomb calorimetric methods, as will be related in the section on energy changes in embryos. Gayda had expected that he would find the total amount of heat given out to be the same at all temperatures, for, as Chambers and Terni had shown, the amount of growth in frog larvae was the same, only metamorphosis supervened earlier at the higher temperatures than at the lower ones. Finally, Gayda compared the heat production of the eggs with adult frogs. The latter evolve 0-45 gm. cal. per hour per gram on an average, as against the maximum of 0-98 gm. cal. per hour per gram at the 20th day from fertilisation.
4-9. Respiration of Insect Embryos
The first organism of this kind which was examined was the silkworm, Bomhyx mori. Apart from early work by Duclaux, the first papers were those of Luciani and Luciani & Piutti, who estimated quantitatively the gaseous exchange of the eggs. The silkworm embryo has a complicated course to pursue, for the egg is laid in the late summer or autumn, and the first week after fertilisation is passed before the hibernation period can be said to have begun. During this time the colour changes from pale yellow to greyish brown. Throughout the winter the egg remains in a sort of latent state, but when the spring begins the developmental process is suddenly released, and 1 1-14 days are sufficient for hatching. A similar quiescent period or "diapause" is observable in grasshoppers and may be shortened or lengthened, according to Bodine, by raising or lowering the temperature. At high temperatures the quiescent period may only be represented by a trough but it cannot be abolished altogether^.
Luciani & Piutti were not able to confirm the preliminary results of Luciani himself, that during the hibernatory period the eggs could li\ e without oxygen, but found instead that the respiration at that time was directly proportional to the partial pressure of oxygen.
^ Conditions in orthopteran development are rather complicated, and for the details reference should be made to the memoirs of Bodine. Respiratory Quotients of 07 to 0-8 seem to be usual in this material.
688
THE RESPIRATION AND
[PT. Ill
O Luciani S^Piutti ♦ Farkas (i) O „ (2)
Excessive amounts of oxygen exerted a marked toxic action. In normal development (after the winter diapause was quite passed through), the amount of carbon dioxide given out per day per kilo of eggs rose quite regularly until at hatching the value was 259 times what it had been initially. In Fig. 136, constructed from the data of Luciani & Piutti, the graph of this process is given ; it does not, of course, represent metabolic rate, for nothing is known of the weight of protoplasm present at different stages. During the whole period 55-1 18 gm. of carbon dioxide were given off by i kilo of eggs, representing a loss of 1-5 per cent, of the carbon originally present. Throughout the whole period, including the time of hibernation, the behaviour of the eggs as ,,_ regards weight was variable, for sometimes they became ^ heavier, owing to absorp- ^ tion of water in a humid oatmosphere, and sometimes gg they lost weight, owing to s exhalation of water in a dry "^ atmosphere. Physiologically '^ they were apparently un- > affected, except that more > carbon dioxide was evolved q weight for weight during the ^ wet periods than during the S dry ones^
The respiratory quotient gave a curious result, for while it was about 0-97 at ^^' ^^ '
the beginning of the developmental period it then rose steadily, passing unity when between a third and a quarter of development had been completed, and rising to 1-305 by the time of hatching. Luciani & Piutti considered that carbohydrates were being combusted throughout development.
Some of the less important conclusions of Luciani & Piutti were
Days of development after the end of hibernation
1 Ashbel, in later work, obtained curves very similar to Bodine's, showing first a somewhat intense respiration, which dies away after 4-5 days, giving place to the quiescent period's almost imperceptible gas-exchange. The silkworm egg respires much less before than after fertilisation (see p. 640) and gives out a gas, probably CO2, for some time after laying, even when not fertilised (see pp. 712 and 819).
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
689
confirmed by Quajat in 1899, who, however, made no measurements of the respiratory quotient, and in 1903 Farkas went into the matter again. He used the same technique as that of Bohr & Hasselbalch on the chick, and his experiments did not begin until about a fortnight before hatching, at which time the sudden rise in the respiration occurs. Farkas' figures were in good agreement with those of Luciani & Piutti; thus in his experiments i kilo of eggs just before hatching evolved 8-7 gm. of carbon dioxide per day,
Days Leptinotarsa decemlineata
4-0
1
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2-4 C 2-0
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6 S 1.2
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Days
Anasa Irisiis
Fig. 137. {The upper solid line in each figure represents the 0^ intake, the lower solid line the CO^ output, and the dotted line the R.(l-)
while in theirs i kilo of eggs at the same time evolved about lo gm. A curve constructed from his data is given in Fig. 136. Unfortunately, he did not make any determinations of the oxygen uptake, so no respiratory quotient was calculated.
In 1925 Fink made a careful examination of the respiratory exchange of 10 different kinds of insects, mostly beetles, using the Krogh differential manometer and Jacobs' modification of the Haas colorimetric method for studying carbon dioxide elimination. Figs. 137 and 138, taken from Fink's paper, all show the curves obtained for carbon dioxide output during the embryonic development of beetles. In each case there is what Fink calls a preHminary "formative period", followed by a continuous rise in respiration (grams of
690
THE RESPIRATION AND
[PT. Ill
carbon dioxide excreted per gram egg per hour — therefore not metaboHc rate) . In Crioceris asparagi, Hylemyia chortophila, and Leptinotarsa decemlineata (the asparagus beetle, the seed-corn maggot, and the potato beetle respectively) the formative period is evidently very brief, not occupying more than a single day, but in Popillia japonica (the Japanese beetle) it lasts for more than 6 days, and, when the respiration does rise, it rises very steeply. The formative period mentioned by Fink perhaps corresponds to the early flat part of the curve in the case of the measurements on the silkworm egg by Luciani and Farkas, but, as in nearly all cases (e.g. the chick)
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123456 789 10 11 Days
Popillia japonica
1 234 5 6 789 10 11 12
Days
Cotinis nitida
Fig. 138. [The upper solid line in each figure represents the Oj intake, the lower solid line the COo output, and the dotted line the R.Qj)
respiration rises in a curve convex to the abscissa, it is questionable what definite meaning can be attached to the "formative period". Fink suggested that there was a correlation between short formative period and the deposition of eggs on foliage or soil surface (examples would be Leptinotarsa and Hylemyia) on the one hand, and between long formative period and deposition of eggs at some depth below the surface of the soil (examples : Popillia and Cotinis nitida (the green June beetle)).
Fink drew a very interesting comparison between embryonic respiration and the respiration in metamorphosis when he set side by side the carbon dioxide and oxygen turnover per gram per hour during the two processes.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 691
Table 80.
Mgm. carbon dioxide out
Mgm. oxyger
I intake
put per gram per hour
per gram per hour
Egg
Pupa
Egg
Pupa
Leptinotarsa decemlineata ...
... 4-89
3-43
4'05
1-82
Hylemyia cilicrura ...
... 12-30
7-89
—
Popilliajaponica ...
... 18-90
3-87
3^5
—
Hippodamia convergens
...
i"57
Epilachna borealis ...
11-00
5-10
1-67
Crioceris asparagi
—
3-46
2-72
Cotinis nitida
—
— .
3-27
Macrocentrus ancylivora
—
—
3-17
Ancylis comptana
—
—
—
5-IO
It is evident that with one or two exceptions the intensity of respiratory exchange is much greater in the egg than in the pupa, so that this at any rate marks a definite difference between embryonic development and metamorphosis. The respiratory quotients were always found to be low. Thus Anasa tristis (the squash bug) gave an initial value of 0-521 rising on the third day to 0-732, and maintaining itself at that level through subsequent development, while the respiratory quotient oi Leptinotarsa ranged daily from 0-512 to 0-68. Cotinis nitida went even lower, seldom rising above 0-524, and even dropping for several days to 0-413. Popilliajaponica began with a respiratory quotient of about that level, but rose in the last few days of development to 0-732. No explanation is available for these curious quotients; evidently either an abnormally small amount of carbon dioxide was escaping or an abnormally large amount of oxygen was being taken in. There seems no reason, from a technical point of view, to doubt the accuracy of Fink's analyses, so it is probable that in these insects whatever combustion processes are going on are obscured by transformations of another order, such as the passage of fat into carbohydrate. This increase of highly oxygenated material at the expense of less oxygenated material leads, in the well-known case of the hibernating marmot, to very low respiratory quotients, and might perhaps be due in the case of these beetles to the formation of chitin.
Melvin later estimated the oxygen uptake during the embryonic development of a number of insects, using the Krogh micromanometer. The organisms employed were Anasa tristis, the squash bug; Tropoea luna, the luna moth; Samia cecropia, another moth; and Pyrausta ainsleii, the smartweed borer. By a method not stated, Melvin was able to measure the weight of the shells of the eggs and
692
THE RESPIRATION AND
[PT. Ill
this he deducted from the weight of the whole egg in his calculations — it amounted to an average of 25 per cent., for the exact figures see p. 322. The oxygen consumption expressed in cubic milhmetres of oxygen per gram egg-contents per hour rose very steadily throughout the incubation period, and this, of course, is exactly what was found by the workers on the silkworm egg. It gives us no information about the metabolic rate of the embryonic cells, and is simply a reflection of the increase of organised living matter with corresponding decrease of the non-respiring yolk. Melvin expressed disagreement with Fink's hypothesis of the formative period in relation to foliage eggs and earth eggs, and his results certainly do not support it. He also made the interesting observation that temperature had almost no effect on the respiration at the beginning of development though it acted powerfully at the end. Thus a rise of 20° in the external atmosphere gave a rise of o-oi mgm. oxygen per gram per hour on the ist day of incubation and a rise of 1-92 mgm. oxygen per gram per hour on the last day.
4-10. Respiration of Reptile Embryos
Only one investigation has been made of the respiration of the reptilian egg. Bohr in 1904 worked on the snake. Coluber natrix, from this point of view, having been instigated to do so by the results he had already obtained on mammalian and bird embryos, and by the researches of Pembrey, Gordon & Warren on the development of heat regulation in the chick. These snake's eggs were developing under natural conditions in a heap of leaves at a temperature of about 29° and in an atmosphere which, on examination, turned out to have only 47 per cent, of oxygen and as much as 13-8 per cent, of carbon dioxide. Bohr was able to incubate them artificially by keeping the air very humid, though he did not attempt to reproduce an atmosphere of that composition. His figures are shown in Fig. 139 graphically. About three
g;0-320 Z 0-310 g 0-300 2.0-290 "S 0-280 I 0-270 Z 0-260 f 0-250 '^0-240 8 0-230 0-220
10 20 30 40 50 60 70 80 90 100
Time, hours Fig. 139
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 693
times as much carbon dioxide was given out at 28" as at 14°, so that the Q^^q for respiration in this egg closely approached that for developmental rate (see p. 508). The metabolic rate values worked out as follows :
C.c. carbon dioxide per kilo per hour
Weight of embryos
At 28°
\ ^
At 15°
0-38 o-8i
659 467
150
1-40 After hatching (3-8)
362 240
90
SO that there was a very obvious decline in the respiration intensity with increasing age. In view of the fewness of the figures, no stress can be laid on the shape of the curve, but it does not seem to follow the course suggested for metabolic rate curves by Murray, i.e. slow decline at first, followed by greater rapidity of fall. The table shows also that the rule of decline appHes to eggs incubated at 15° as well as at 28°. Here we have a case like that of the chick, where the earliest stage which it has so far been possible to examine has the highest metabolic rate of all. If figures could be obtained for the snake or the chick at about the time of gastrulation a great advance would have been made. The respiratory quotient of the snake's egg was shown by Bohr to remain in the close neighbourhood of 0*9 throughout development, so that he concluded there was a dominant complete combustion of carbohydrate material.
4-11. Respiration of Avian Embryos in General
I have spoken already about the earlier researches on the respiration of the avian embryo. The modern period in this subject begins in 1900, when Bohr & Hasselbalch published the first of their series of classical papers on the evolution of carbon dioxide, the absorption of oxygen and the heat-production of the hen's egg. Their first paper was concerned exclusively with the production of carbon dioxide during the incubation period, for they wished to study the relation of metabolic rate to age and weight, a correlation the importance of which none of the earlier workers, such as Baumgartner or Pott & Preyer had appreciated. Their apparatus consisted of a thermostat chamber in which the egg was placed connected with a chain of absorption-tubes, etc. Experiments with empty egg-shells from
694 THE RESPIRATION AND [pt. iii
freshly laid eggs showed them that an appreciable amount of carbon dioxide could be given off from bicarbonates in the shell — a finding that might have been expected in view of the fact that the oviduct of the hen is probably saturated with carbon dioxide, unlike the air outside. Compare with this result the work of Bialascewicz & Bledovski on amphibian eggs^. These experiments with shells alone provided Bohr & Hasselbalch with a correction which they introduced into the values obtained for fertile normally developing eggs. This correction, while negligible in later stages of development, was very important in the early stages where the respiration of the embryo is small. More often they got over the difficulty by keeping fertile eggs at room temperature in a current of carbon-dioxide-free air until they gave ojff no more of the gas. Controls carried through for 3 weeks on infertile eggs showed that Pott & Preyer had been wrong in their conclusion that infertile eggs gave off notable quantities of carbon dioxide, for, on the contrary, the amount given off was remarkably small, varying from o to 5 mgm. per 24 hours. Then they proceeded to the experiments with the developing embryos, obtaining the diagram shown in Fig. 140. The varying width of the columns is a measure of the length of time the experiment was conducted ; thus in one or two cases, it was as much as 24 hours, but in the majority only 4 or 5. A glance at the graph shows the initial production of gas from the shell, the steady rise from the 2nd day onwards, and the plateau which Bohr & Hasselbalch always got from the 17th to the 21st day. Their next interest was the variation in the respiration intensity. For this it was necessary to make weighings of the embryos, for at that time the fragmentary values of Falck were all that were available. The numerical results which Bohr & Hasselbalch obtained are shown in Appendix i, and agree well enough with those got by later observers, but they made the interesting correlation that the curve for total weight (not weight increments) went up in exactly the same manner as the curve for the carbon dioxide excretion in cubic centimetres per 24 hours (i.e. increments of respiration) . This is shown in Fig. 141 taken from their paper, and it should be noted that the agreement is rather better after the 9th day than it is before it. The metabohc rate values are shown in Fig. 142 constructed from their data. A sharp descent brings the metabolic rate down to what is
mgr. C02 given off per hour per egg. 31
9 10 11 12 13 U 15 16 17 18 19 20 21 D. Days
Fig. 140.
70
60
50
40
30
20
10 300
90
80
70
60
50
40
30
20
10 200
90
80
70
60
50
40
30
20
10 100
90
80
701 60
50
40
30
20
10
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rk--r 1 1 1 . 1 1 i 1 . 1 1 1
J 1 — 1 —
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0.
Fig. 141. CO2 weight
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
697
o CO2 (Bohr&,Hasselbalch1900) ® ^^sUhasselbalch 1900) • Cbj (Murray 1926)
practically the adult level (Regnault) by half-way through the incubation period, but Bohr & Hasselbalch's weighings were few in number, and it would not be just to lay much emphasis on the shape of the curve. That it descends so markedly, in opposition to the ascending curves of the earliest stages in echinoderms and amphibia, for example, is all that need at present be stressed.
In the second paper Hasselbalch went on to investigate the oxygen consumption of the eggs, and to calculate the respiratory quotient. This last had only previously been approached by Baumgartner, who, having found that i -63 litres of carbon dioxide were given off during the whole of incubation, and that 1-76 litres of oxygen were taken in, concluded that the average respiratory quotient |s was 0-93. Pott & Preyer's re- a^ spiratory quotients had been f obviously wide of the mark, ^^ reaching in some cases 3-59. I3 Hasselbalch devoted a long a preamble to the shortcomings >^ of the earlier investigations, 5i and first paid attention to the 8 growth-curve of the chick em- " bryo, adding more figures to those of Bohr & Hasselbalch, which are given in Appendix i. He made measurements also of the relation between the embryo and its membranes, and of the water-content of embryo and allantois — these are mentioned elsewhere in their proper place.
Controls on infertile eggs showed that only an extremely small amount of oxygen was taken up by eggs without living embryos, not more than 0-15 c.c. per hour. A slight escape of nitrogen from the eggs seemed to occur, but was very insignificant in amount. Hasselbalch then went on to experiments with fertile eggs. The oxygen consumption per hour followed the carbon dioxide output closely, and rose in the same way as the weight curve, just as Bohr & Hasselbalch had found to be the case for the increments of carbon dioxide production. The respiratory quotients found varied round about 0-7, but discussion of them will be deferred for a moment. Hasselbalch found that, during the entire incubation
45-2
Tf-^-^-a ^
^
9 10 11121314 15 16 17 13 19 20
Days of development
Fig. 142.
698
THE RESPIRATION AND
[PT. m
period, 3-0225 litres or 5-939 gm. of carbon dioxide were produced, so that, calculating from an average respiratory quotient of 0-677, 4-465 litres or 6-384 gm. of oxygen was used, and that value was, indeed, very close to the one experimentally found. Hasselbalch reckoned that, as the 5-939 gm. of carbon dioxide given off corresponded to 1-620 gm. of carbon, and as, according to Liebermann, egg-fat contained 71-67 per cent, carbon, 1-620 gm. of carbon accounted for 2-260 gm. of fatty acids. This was in very encouraging agreement with Liebermann's figure, calculated from chemical analysis, that 2-762 gm. of fatty acids disappeared during development. It was not unnatural that Hasselbalch should conclude that fatty acids were the sole source of embryonic energy during incubation, though to do so was certainly to forget the presence of protein breakdown-products in the allantoic fluid, so obvious in the egg, and to take insufficient account of the intervention of more complicated processes than the simple oxidation of fatty acids to carbon dioxide and water. From his oxygen figures, Hasselbalch went on to calculate the metabolic rate, which worked out in good confirmation of the rates reported in the previous paper, and is shown in Fig. 143. The weight of the membranes is not included in this calculation, and until some information concerning their respiratory intensity is available, their influence on the metabolic rate curve cannot be assessed (cf the data of Bycrly in Appendix i). Hasselbalch concluded as the result of his experiments that an exceedingly small amount of a gas other than carbon dioxide was lost by the egg during its development, and believed it to be nitrogen. He returned to this puzzling phenomenon in a subsequent paper in which he again asserted its objectiveness, maintaining that 0-5 c.c^
8000
"\
7000
\
Metabolic rale (oxygen)
6000
® Danish O2
fe \
• Murray O2
5000
C Y
s: \
4000
3000
- \
5
2000
5
^--^tr^
1000
^^^^-^t:^::^^^
1 1 1
, , , i^^^", 1 1 1 , . > ,
3 4 5 6 7 8
121314 1516 17 1819'
Fig. 143.
i
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 699
of oxygen and 2-0 ex. of nitrogen were given off by fertile eggs during the first week of incubation. He tried many methods in his efforts to get to the bottom of it; thus he studied the gaseous exchange of the whole yolk in vitro, the composition of the air in the air-space, and of that extractable from eggs by bringing them into a high vacuum. His results left him with the conviction that there was an output both of oxygen and nitrogen, and he suggested various possible mechanisms which would account for it. Typical experiments are shown in Table 81. It is probable that they simply represent the
Table 8 1 . Hasselbalch's experiments.
Cubic centimetres of gas
Time of the
Carbon dioxide
Nitrogen
Oxygen
^
ment
Taken
Given
Taken
Given
Taken
Given
in hours
up
off
up
off
up
off
Fertilised egg, ist day
5-0
—
0-09
0-36
0-44
>, ,,
5-0
—
0-I5
—
078
—
0-24
,, ,,
5-0
■ — •
0-05
—
1-40
—
0-40
Fertilised egg, 2nd day
4-5
^
0-04
—
GIG
G-04
—
Unfertilised egg
5-5
0-34
—
—
1-47
—
0-85
„
4-0
0-02
—
—
G-88
—
039
G-6l
,,
4-5
—
0-37
—
2-37
—
,,
4-0
—
0-09
—
I -06
—
0-35
Fertilised yolk in 0-82
4-5
—
006
—
0-13
—
G-l6
% saline
5J
6-25
o-io
—
—
o-ii
—
013
11 ).
4-0
None
None
—
0"34
G-20
Fertilised yolk in 0-59
4-5
—
0-13
—
None
—
0-13
% sodium fluoride
Unfertilised yolk in
4-0
0-05
—
001
—
—
g-g6
0-82 % saline
,, ,,
6-0
—
o-io
o-i6
—
—
gg6
adjustment of the shell, the yolk, and the white, as they gain gaseous equilibrium with the external air. Hasselbalch unfortunately did not state how long a time elapsed between the laying of the eggs and his respiratory experiments upon them. In spite of the fact — plain from his tables — that unfertilised eggs gave off as much oxygen as fertilised ones, he persisted in maintaining that "the condition for physiological oxygen-production in the first hours of development is the presence of living cells". By means of his in vitro experiments with yolks in salt solutions he showed that the osmotic pressure had an influence on the gases given off; thus hypotonic solutions (i per cent, sodium fluoride, etc.) led to a decrease in the oxygen generated, but hypertonic solutions set up oxidation processes in the yolk which abolished it altogether by causing an oxygen uptake.
700 THE RESPIRATION AND [pt. iii
"The oxygen may be", said Hasselbalch, "either a by-product of syntheses associated with cell-division — possibly of fundamental nature, analogous to the Og-assimilation of green plants, and normally obscured by the mass of respiration — or on the other hand, it may be purely a side-issue, originating from some fermentative process or other associated with growth." And there is, as we have seen, a third possibility.
Brandes has more recently discussed the question afresh and regards the oxygen given off by the yolk in Hasselbalch's experiments as of much importance for the life of the embryo, cut off as it is from the air by the thick shell and the mass of albumen. His remarks do not include any chemical explanation of its origin but as we shall see in Section 14-6 the yolk of the avian egg contains catalase in an active condition and it is legitimate to suppose that the concentration of hydrogen peroxide, activity of the enzyme, etc., may be so arranged that an appreciable part, if not all, of the oxygen requirements of the embryo during the first day or two, is provided for in this manner. Brandes divides the respiratory life of the chick embryo into the following stages :
A. A "molecular" respiration of yolk-oxygen
(i) without the presence of haemoglobin (i day),
(ii) with the presence of haemoglobin (2nd to 19th day),
(a) by the blastoderm circulation (up to the 6th day),
(b) by the yolk-sac circulation (up to the 19th day).
B. A respiration of atmospheric air
(i) through the allantoic vessels (from the 5th day onward), (ii) through the lungs (from the 1 7th day onward) .
Brandes called attention to the old work of Dulk in this connection. It is doubtful whether stress can be laid on the results obtained with the technique of 1 830, but nevertheless it is interesting to recall that the gas which Dulk obtained from whole eggs contained 6 per cent, more oxygen than atmospheric air and, after 10 days' development, li per cent. Bulk's work has already been referred to on p. 617. His figures were:
% oxygen
Air 208 -2I-I
Gas from whole eggs (o days) 25-26-26-77
,, 5, (10 days) 22-47 (with 4-44 % carbon dioxide)
,j ,, (20 days) 1 7-9 (with 8-48 % carbon dioxide)
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 701
In one respect, at any rate, Hasselbalch's experimental findings were subsequently reversed, for a few years later Krogh, who was undertaking an extended test of the question whether animals excrete small amounts of nitrogen or not, took occasion to examine the hen's egg from this point of view. His experiments, which were conducted with irreproachable technique, led to quite negative conclusions. For the most part he occupied himself with later stages than Hasselbalch, but even on the ist day of development he could find no evolution of nitrogen which was outside the small experimental error. His conclusion was that certainly not more than 0-003 c.c. per hour, or 2-5 mgm. of nitrogen for the whole 3 weeks of development, was excreted, and so far the 2-5 mgm., if indeed they do leave the egg, have not been missed by chemists. Krogh's experiments were fully supported by some work of Tangl & von Mituch. "Hasselbalch found", said Krogh, "by evacuating egg-contents in the mercury pump, that fresh eggs contain a considerable surplus of dissolved gases above that which could be taken up by a corresponding quantity of pure water. The surpluses are, according to him, confined to the yolk and I venture to suggest that it is the fatty substances which dissolve the gases. In an egg of 60 c.c. about 1-2 c.c. of nitrogen is contained whereas 60 c.c. of water saturated with air at 38° contain only 0-55 c.c. Hasselbalch found that after two days' incubation the surplus had sensibly diminished and there can be no doubt that the whole of it will be given off during development as the substances of the chicken dissolve less of the gas than pure water."
Pott had claimed that much more carbon dioxide was given off by an egg in pure oxygen than in ordinary air, but his technique was inferior. Hasselbalch found that whatever the effect was, it was very variable; thus in one experiment in 82 per cent, oxygen the carbon dioxide output was half the normal, and the oxygen uptake only a quarter the normal, but in 79 per cent, oxygen the carbon dioxide output was slightly raised, while the oxygen uptake was three times the normal. These effects could only be due to toxic action of high oxygen concentrations (see Riddle's work in Section 18-9) alternating with true accelerating effects. Hasselbalch also concluded that the excretion of gaseous nitrogen from the eggs could take place at these high oxygen concentrations to a far greater extent than under normal conditions, as much as 2-268 c.c. being given off per hour in one experiment. Krogh's work, which did not
702 THE RESPIRATION AND [pt. iii
include data for abnormal oxygen concentrations, cannot help us here, and Hasselbalch's observations on nitrogen excretion still remain mysterious.
The observations of Bohr & Hasselbalch on the heat production of the hen's egg were published in 1903. They knew the amount of oxygen used and carbon dioxide excreted, and the amount of fat
Table 82. Respiratory quotients of Chick.
Calculated by
Needham from
Days'
Given by
the figures
develop
Given by
Bohr &
of Bohr &
Given by
Given by
ment
Lussana
Hasselbalch
Hasselbalch
Hasselbalch
Murray
I
—
—
—
0642
—
2
—
—
I-OIO
1-318
—
3
—
—
0-960
0341
—
4
(0-6971
o-8oi
5
—
0-890
0-673
—
6
—
—
0-6x9
0-653
0-60
7
f 0-5601 1 0-600
o-6o6
0-69
8
0655
f 0-530) 1 0-500/
0-710
0-75
9
\tm
0-500
0-679
0-79
10
/0-7471 IO-703J
0-500
0-706
0-81
II
fo-490) 1 0-450 j
0-734
082
12
—
0-751
0-300
0-628
0-81
13
f 0-646) 1 0-712)"
i-ooo
0-685
0-79
14
0-533
(0-705) I0-675I
—
0-669
0-76
15
0-527
o-68i
0602
0-647
0-72
16
0-514
fo-735 i I0-679)
—
0-716
0-70
17
0-614
0-708
—
0-678
0-69
18
0-630 0-761
0-718
—
0-657
0-70
19
0-693
—
0-716
0-71
20
—
—
0-675
—
21 o-68o — — • — —
burned. They rightly regarded it as very important to know whether the energy of the fatty acids could all be accounted for as heat put out during development, and, if not, what proportion of it could be. With this aim in view they constructed a differential calorimeter in which they were able to incubate single eggs and examine the heat production of them at the same time as their gaseous exchange. The figures they obtained were very numerous. Attention may first be directed to the respiratory quotients, which are collected together in Table 82 and in Fig. 144. Five series are available, those of Bohr
)
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 703
& Hasselbalch, those of Hasselbalch, those calculated from Bohr & Hasselbalch's data, those of Lussana, and those of Murray, whose papers will be mentioned below. As Fig. 144 shows, there are a certain number of points above 0-75, although the greater number lie just below that figure. What is probably significant is that the latter occur mostly after the 8th day of development, while the
G>
100
09
8
07
© R.Q. calculated from chemical analyses © Experimentally determined by Bohr and Hasselbalch and given by them Q ditto, but calcd. from their figures by Needham
O Experimentally determined
by Lussana
O Corrections for alkali
reserve
06
I
5 10
Days Fig. 144. former all occur before it. No high point is to be found after the loth day^. Lussana's points agree well with those of Bohr & Hasselbalch and Hasselbalch. Of the high respiratory quotients, the only point actually given by Bohr & Hasselbalch was 0-890 for the 4th day; the others were all calculated from their data in 1927 by me. At the same time I also attempted to see to what extent the alkali
1 Dickens & Simer give the R.Q. of 5th day chick embryos in vitro (phosphateRinger, pH 7-4, 38°, with 0-2 % glucose) as unity.
704 THE RESPIRATION AND [pt. hi
reserve of the egg-contents would affect the values obtained for gaseous exchange, for this source of error was neglected altogether by the Danish workers. From the careful investigations of Aggazzotti on the pH, and of Healy & Peter on the total acidity of the yolk and white, I made an estimate of the extent to which the respiratory quotient values would be affected if carbon dioxide were retained and neutralised instead of being excreted. The results, shown in Fig. 144 by special points, showed that the values might come higher by as much as 0-5 respiratory quotient units when so corrected, but not more. The effect of the alkali reserve may therefore be regarded as quite small. The line joining the series of points in Fig. 144 represents the respiratory quotient calculated from various chemical analyses; this will be referred to in detail later. On the whole, the evidence points to a combustion of fat after the 8th day (Bohr & Hasselbalch's average then was o-68) and to more complicated events before that time.
4- 1 2. Heat-production of Avian Embryos
Bohr & Hasselbalch's observations on the heat-production of the egg are shown graphically in Fig. 145. There are several remarkable things about this curve. Firstly, during the first few days of development they observed an absorption of heat, not an output. They were convinced that this process could not be accounted for as a meaningless effect due to technique, but that it was a real physiological phenomenon, and they associated it with the production of oxygen which Hasselbalch had previously shown to go on before the 3rd day. They pictured the existence of some endothermic synthetic process which gave off oxygen as a by-product, and even suggested that this might go on throughout development obscured by the mass of the usual respiratory exchange. No satisfactory explanation has so far been advanced for the initial heat absorption shown in Bohr & Hasselbalch's work, and, as no calorimetry of the egg has since been published, it has remained unconfirmed, although Barott, I understand, has unpublished experiments indicating that it is an artifact. Its possible theoretical importance has already been indicated in connection with the work of Rapkine on the echinoderm egg (see p. 648). The second striking thing about the figure is the fact that the observed values for heat-production agree so well with the values calculated from the oxygen taken in and the carbon dioxide
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 705
put out, on the assumption that the combustion is going on at the expense of fat. The observed and calculated points lie for the most part on exactly the same line. Table 83, which gives the average values for observed and calculated calories evolved per hour on each day, shows up the divergences better. After the early period of heat absorption, there follows a second period in which the observed values are somewhat lower than the calculated ones but by the nth day equivalence has been gained, and, in the third period,
100
90 80 70 60 50 40 30 20 10
10 20
Bohr 8^ Hasselbalch (heat production)
O Observed © Calculated
J L
J \ L
J 1 L
J \ L
J I
10 11 12 13 14 15 16 17 1819 Days
Fig. 145.
when the heat radiated is reaching high figures, the advantage is rather on the side of the observed values. Bohr & Hasselbalch noticed this fact, and, by a comparison of the percentage differences in each case, showed that they cancelled out almost exactly over the whole time of incubation, the earlier lag on the observed side making up for the later lag on the calculated side. Thus for the entire incubation period 12-16 kilo cal. were experimentally found in the calorimeter, while 1 2- 1 1 kilo cal. were calculated as the expected amount — an excellent agreement. The extreme importance of these experi
7o6 THE RESPIRATION AND [pt. iii
merits will appear in the section on Energetics and Energy Sources of the embryo. For hourly radiation the correspondence was equally good, being 506-8 gm. cal. observed and 504-72 calculated. Thus there was no energy unaccounted for, none held back for the purpose of maintaining the embryo in physico-chemical equilibrium, or as "Entwicklungsarbeit". "Wahrend der Entwicklung des Embryos", as Bohr & Hasselbalch put it, "in bedeutenden Mengen
Table 83. Heat-production of chick embryo in gram calories per hour produced.
Averages
Observed
Calculated from
the respiratory
Days
Original data
Smoothed
exchange
I 2
-0-77
-1-50
_
3
-1-47
-0-90
—
4
0-39
0-39
2-52
I
340
2-00
2-84
2-63
3-22
37§
I
5-"
4-55
5-86 6-62
8-42
5-27
9
6-69
8-56
10
10-19
10-20
II
17-27
17-10
17-77
12
2463 3538
24-63
24-85
13
32-50
30.76
14
41-62
41-62
42-97
15 16
51-64 60-14
51-64
53-24
61-50
59-48
17
86-02
73-00
73-18
18
87-06
82-50
81-45
19
90-07
90-07
89-75
umgesetzten chemischen Energie auf neugebildete Gewebe nichts iibergefiihrt wird, dass dieselbe dagegen in ihrer Gesammtheit das Ei als Warme verlasst." Tangl's earlier papers were appearing at this time, and they naturally found in his estimations of the calorific value of the egg-substance at different times during development confirmation of their view that fatty acids were the exclusive source of energy by combustion. It is only fair to add that they did take into account the possibility of combustion of proteins, but their arguments against it were extremely poor. From their figures for heat-production, Bohr & Hasselbalch did not calculate the metabolic rate by referring them to i gm. of embryonic body- weight, probably because they were doubtful about how much the membranes might be producing. The results of such a calculation^ are shown in Fig. 146,
^ Leaving the membranes out of accoimt.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
707
)(4 days) 46-6 ) 18-3
Heat production (metabolic rate)
Heat B.S^H. wt. B.&H,&H.
HeatB.aH.wts. M.
Calcd. by Le Breton &,
Schaeffer from B.SiH.
from which it can be seen that the fall is most pronounced, the metabolic rate being above 15 gm. cal. per gram on the 5th day and below 4 gm. cal. per gram on the I gth day. The significance of the kink in the curve at the gth day is obscure. As the curve in which Murray's weight measurements are used shows it too, it must be due to the heat data. On the whole, a good resemblance can be traced between the heat-production metabolic rate curve and the gaseous exchange metabolic rate curves shown in Fig. 143, for in both cases the low value characteristic of the end part of incubation is attained about half-way through development or slightly before. The question of re- ^^" '^ lating the heat-production to the surface of the chick embryo, which is rather a complicated one, will be left until the section on Energetics. If now, aided by the investigations of Bohr & Hasselbalch, we enquire what are the number of ^ gram calories produced | during each period of % weight doubling, we find | there is first a fall and > then a rise. In Fig. 147, I which has been plotted e from such a calculation, I the times required to | double the weight at dif- ^ ferent ages are given along one ordinate, while the
Days
12 13 14 15 16 17 18 19
C70
y
yC"^ — ®
.60
"
y^ 0/
<L)
^
Z'
D /
5 50
13
"<
°x
/
\
/
■§
\
y /
o40
>ii
/ /
a
"v
^ / /
-a
y%^~±^
(U
y
.h30
oX
cr
/
• -1 Fig- 150 but that figure is certainly too
high. In direct bearing upon these questions was his calculation of the respiratory quotient. According to him, it varies during the period under discussion from 0-82 to o-6o. Curiously enough, his lowest value, o-6o, he obtained on the 6th day, i.e. the very time when a particularly high one would have been expected. However, if his oxygen consumption on that day was in error on the high side, as has already been surmised, then the true respiratory quotient for that moment would be much higher.
Apart from the work of Warburg and his collaborators, which will be treated as a whole below. Shearer is the only investigator who has
6 17 18 19 20
7i8
THE RESPIRATION AND
[PT. Ill
examined the in vitro respiration of chick embryo tissues. His experiments have already been discussed in relation to Child's theory of metabolic gradients. If reference be made to Fig. 96, it will be seen that the work led to two conclusions, firstly, that the respiratory rate of the head fragments was greater than that of those from the tail, and
'calcL-latedfO B.^ H.heab, Murray 0^
by <© """ " ^Hasselbalch O2 O.N. (^ „„„ „ jShearer02(invifcra) Q Calculabed byCahn
2 13 14 15 16 Fig. 157 secondly, that in both cases the rate declined as development proceeded. Reasons have already been given for doubting the significance of the first of these results, but the second remains unaffected, and adds another piece of evidence to the well-established belief that in the chick embryo metabolic rate declines with age. An amount of tissue containing 2-8 mgm. of nitrogen takes up oxygen as follows:
Day Oxygen in c.mm.
4 18-50
5 12-50
6 5-80
7 302 10 105
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 719
Two attempts have been made to calculate calorific quotients for the chick embryo since the publication of exact data for the heat production and the oxygen consumption. The results of my attempt to do this are shown in Fig. 157, where calorific quotient is shown plotted against the age (a) using Bohr & Hasselbalch for heat and Hasselbalch for oxygen, and (b) using Bohr & Hasselbalch for heat and Murray for oxygen. In both cases there is a fall to the 9th day followed by a rise lasting approximately for the rest of the incubation period, but a glance shows that the values are grossly removed from the theoretical. The dotted line drawn between the horizontal lines shows the course that would theoretically be taken by the calorific quotient supposing that carbohydrate was first burned, then protein, and finally fat. As can be seen, the experimental curves do more or less follow that rhythm, but little weight can be attached to such a correspondence in view of the kink on the heat-production curve (see Fig. 146), which almost certainly is responsible for the drop in the calorific quotient at the 9th day. Nothing would be more welcome than a redetermination of the heat-production curve of the chick embryo, for we should not only then have a better idea whether the kink in question is real or not, but also with improved calorimetry less heat would be lost and the calorific quotient would probably fall within its proper limits. Cahn's views on these questions are discussed in the section on the Energetics and Energy Sources of the embryo.
4-14. The Air-space and the Shell
No mention has so far been made of the recent work on the airspace, a structure present in many kinds of eggs, but occupying a specially prominent position in the case of the chick. Its origin in birds' eggs is obscure, but, according to Lataste, no air-space is present before laying, and its appearance is only due to the contraction of the egg-contents from the rigid shell as the egg cools after leaving the parent body, Lataste supports this view by adducing the fact that eggs with flexible coverings never have air-spaces (e.g. lizards and serpents), and, in the case of shell-less birds' eggs, which are sometimes laid, no air-space appears, though the envelopes may be a little wrinkled. Indeed, as long ago as 1847, Goste had observed that if a laying hen was killed, the oviduct ligated, removed, and then immersed in a dish of oil, an oil-space formed
720 THE RESPIRATION AND [pt. iii
instead of an air-space as the egg and the oviduct slowly cooled. And by clipping a window in the uterine wall, he could produce it at will at any part of the egg. The analogous air-space in the cocoon of the silkworm has been studied by Portier & de Rorthays, who found that carbon dioxide accumulates in it as metamorphosis proceeds, just as during development in the hen's egg. Dubois and Dubois & Couvreur have also studied this air-space.
In the course of his respiration experiments, Hasselbalch investigated the contents of the air-space, obtaining figures as follows :
Days after fertilisation
2
5
Infertile
Hasselbalch was naturally very interested that the fertile eggs seemed to have slightly more oxygen in their air-spaces than ordinary air, in view of his other researches on the oxygen production of the birds' egg during the first few days. The fact that he got normal figures for infertile eggs still further contributed to that conclusion. But the classical work on the subject is that of Aggazzotti, who in 1 9 14 measured the percentage of oxygen and carbon dioxide in the air-spaces of incubating eggs, not only at sea level but at the mountain experimental station of Col d'Olen.
Taking first the normal figures, Aggazzotti found that in fresh eggs the carbon dioxide content of the air-space is high, from 1-42 to 2-05 per cent., while the oxygen content is just equivalent to that of the external air, though it may be very slightly above it (20-72 to 21-29 per cent.). The former fact agrees excellently with the preliminary output of carbon dioxide (due to the oviduct), which so many workers have observed, and perhaps the latter fact may be taken as confirmation of Hasselbalch's statements about oxygen production. After 8 or 9 hours, however, the carbon dioxide has fallen to o-6 per cent., while the oxygen has remained stationary,
C.c. of air
in air-space
Oxygen %
1-25
20-96
0-702
21-35
0-703
21-53
0-479
21-57
1-949
20-65
Average ,
... 21-21
0-32
21-37
0-651
20-74
Average ,
... 20-96
Atmosph
leric air
... 20-96
J
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
721
and, if the egg is infertile, no further change takes place, even though it be kept for a month or more. These alterations are shown in Fig. 158. If the egg is fertile, the carbon dioxide content then rises during the ist day to 1-89 per cent., a phenomenon probably due to the effect of heat on the egg-contents. The subsequent course of the curve is shown in Fig. 159. A slight diminution on the 2nd or 3rd day brings the value to a level at which it remains until roughly the nth day (i-o6 to 0-33 per cent.), but after that time it progressively augments until hatching. Clearly it is at about the nth day that the carbon dioxide produced by the metabolism of the embryo becomes greater per unit time than that which can get away
Unfertile eggs 2-0 '"
Airspace composlbion
- 1-0
5 10 20
Days Fig. 158.
through the egg-shell per unit time, hence an increased concentration in the air-space. At the end of development the percentage has risen to between 4-50 and 5-43. During the first week the oxygen content remains unchanged, and a little below that obtained in unincubated eggs, but after that time it begins to fall, presumably because it is being used up by the embryo rather faster than it can diffuse in through the egg-shell. At the end of development it only reaches the figure of 13*65 per cent. If now the curve in Fig. 159 be compared with that found by various workers for the output of carbon dioxide from the egg (Fig. 151), it can easily be seen that, although the latter increases during incubation more than fifty times, the former hardly increases five times; the obvious inference is that the shell becomes more permeable to gases as development proceeds. It is unfortunate that no direct measurements with a diffusiometer
722
THE RESPIRATION AND
[PT. Ill
have been made of the shell during the development of the chick, but analyses which will be discussed later (see Section 13-2) do demonstrate that there is a definite loss of inorganic and organic substances from the shell, and it is a well-known fact that the shell becomes more brittle as development proceeds. Only one study of the histology of the shell during incubation has been undertaken, namely, that of Rizzo in 1899. Rizzo found that the number of pores per square millimetre of shell surface varied from o-86 to 1-44, with an average of 1-23. A hen's egg has an average surface of 6644 sq. mm., and about 7600 pores. Rizzo's method was to drain the contents of the egg
Ferbi
e eggs
COaCold'Olen
OzCold'Olen 02Norm3l
through two small punctures, after which the egg-shells were carefully washed, and refilled with a weak aqueous solution of methylene blue — the pores were then visible to the naked eye as fine blue points. They were much more numerous over the air-space than elsewhere^. We may conclude that an increased permeability of the shell to carbon dioxide during development is fairly well established and the remarks that have been made on this point apply equally well to oxygen. The loss in oxygen is more or less compensated for by the gain in carbon dioxide, so that the nitrogen content of the air-space remains practically unchanged. Hufner, whose permeability experiments on egg-shells have already been referred to, found that in I second at 11-9° 2-115 c.c. of oxygen would diffuse into the goose's egg (from ordinary air, i.e. a partial pressure of 159 mm.) and 0-503 c.c. of carbon dioxide would diffuse out (to a partial pressure
^ For a physical account of these pores, see Dumanski & Strukova.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 723
of 29-94 mm.). These findings agree well with those of Aggazzotti. Hiifner himself carried out some experiments on the air-space contents ; he was indeed the first worker to contradict the early assertions that there was a much higher oxygen concentration within the air-space than outside. His figures were:
% carbon % oxygen % nitrogen dioxide
Hen 18-94 7997 i-og
Goose 19-71 79-08 I-20
He made an interesting calculation showing that the amount of the gases which could diffuse in was much in excess of those actually found. Baumgartner had stated that, on the 20th day of development, a hen's egg gave off 0-56 gm. (285 c.c.) carbon dioxide and took in 0-44 gm. (310 c.c.) of oxygen. Though the surface of the goose's egg is only four times that of the hen's egg, Hiifner allowed a ten times greater metabolism, but even so found that 3100 c.c. of oxygen and 2850 c.c. of carbon dioxide stood much below the 182,700 c.c. of oxygen and the 43,460 c.c. of carbon dioxide which his measurements would regard as being provided by diffusion. However, Baumgartner's values are very low (60 per cent, of the real values), and the multiplication of those of the hen by 10 to make those of the goose is a hazardous proceeding, so the real gaseous factor of safety of the hen's egg has not yet been calculated, and cannot be until somebody repeats Hufner's observations on the shell of the hen's egg. From some figures of Hufner's, however, for experiments in which the inner membrane had been stripped off, a rough assessment of this can be made in the case of the hen. Here in i second at 9-4° and from a partial pressure of 159 mm. 1-587 c.c. of oxygen would diffuse into the egg over all its surface (surface values being taken from Murray), and the partial pressure being 29-94 mm., 0-554 c.c. of carbon dioxide would diffuse out. This would mean 137,000 c.c. of oxygen and 47,800 c.c. of carbon dioxide per day. As the greatest amount of oxygen taken in is 720 c.c. per day, and the greatest amount of carbon dioxide put out is 510 c.c. per day, there would appear to be an ample margin. But 137 litres seems an immense quantity of oxygen, and it is probable that Hiifner's figures are here far too high; moreover, the inner shell membranes may probably make a considerable difference.
Another interesting point raised by Hiifner was whether the rates of penetration of gases through the egg-shell followed Graham's law,^
724
THE RESPIRATION AND
[PT. Ill
and were proportional to the square roots of the specific weights of the respective gases. All his tables showed that they did not follow this rule, but some more complicated one, being doubtless affected by the complex conditions in the material. Hiifner found, as has been said, that hydrogen penetrated most easily, then carbon dioxide, then nitrogen, and lastly oxygen. The hen's egg-shell was less easily penetrable than the goose's egg-shell, and Hufner suggested that this was necessary for it, since its surface was smaller in proportion to its weight. More recently S. Ancel has shown that the penetration of chloroform vapour into the hen's egg exactly follows Graham's law. Aggazzotti's experiments with eggs incubated 3000 metres above sea level showed (as may be seen from Fig. 1 59) that the composition of the air in the air-space was
almost identical with the nor- '°r Air space increase
91 mal sea-level values. The
percentage of oxygen and of
carbon dioxide was constantly
lower by a small amount, so
that there was a certain degree
of acapnia and anoxaemia of
the embryo, due, of course, to
the fact that at the Col d'Olen
the barometric pressure was
only a third of what it was at
Turin. These high-level experiments have no great value, for all the
embryos incubated at the Col d'Olen died before hatching, and were
more or less abnormal.
No very extensive figures seem to exist in the literature for the change in volume which the air-space undergoes during development, though qualitatively, as is well known, it markedly increases. A curve can, however, be constructed from the data given by Aggazzotti; it is shown in Fig. 160^. It will be remembered that the knowledge of this fact was one of the bases of Mayow's theory of embryonic respiration. The elasticity of the air contained in the airspace acted, he thought, like a kind of piston, compressing the yolk and white into the solid tissues of the chick, but we now regard the expansion of the air-space as the effect of development rather than its cause, and as arising from the evaporation of the water and the
^ Romanov also gives data for this.
J
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 725
combustion of a certain amount of solid. Hanan, in fact, in some unpublished experiments, has noticed a close relation between humidity of environment and size of air-space.
Another interesting corollary of the respiration of the embryo was brought out by Hammett & Zoll. These workers studied the response of the vitelline vessels of the chick embryo to various chemical stimulants, by injecting them into the yolk with a micropipette in the immediate vicinity of a length of vessel observed through a microscope. In this way they ascertained that the walls of the vitelline vessels are sensitive neither to the H ion nor to the OH ion, changes in their concentration varying from pH 5-0 to 9-0 provoking no alterations in the calibre of the vessels. On the other hand they are specifically reactive to carbon dioxide and the invariable response was one of constriction — moreover, the effective agent was not HCO3 but either carbonic acid or carbon dioxide, for hydrochloric acid solutions saturated with the gas were as efficient as water saturated with it^. What follows, or may follow, from these results, was thus suggested by Hammett & Zoll. It is obvious that temperature variations during incubation under the hen would induce variations in the production of metabolites from which the embryo builds its tissues. With rising temperature this would be accelerated and so would carbon dioxide production. The latter by its constrictive action on the vitelline vessels would cut down the blood-supply to the embryo and thus prevent its being flooded with more food than it could profitably handle. With falling temperature the processes would be reversed, and the relaxed vessels, carrying a larger volume of blood of lesser metabolite content, would thus provide the embryo with adequate material for uninterrupted development. It is conceivable that the regulation of the food-supply is controlled in part by the COg-sensitivity of the blood-vessels of the yolk-sac. Hammett & Zoll also applied their views to the process of inclusion of the yolk-sac within the embryonic body at the end of incubation, and suggested that the large amounts of carbon dioxide then being evolved constricted the blood-vessels of the yolk-sac to so great an extent as to cause the atrophy which normally occurs.
^ This was confirmed by Lange. The vascular membranes contain no nerve fibres (Lange, Ehrich & Cohn) and elastic fibres are not to be found in their vessels (Cohn & Lange). The capillaries are more irritable than the arterioles, and at the end of development there is no degeneration ; the vessels die in complete possession of their physiological irritabiliiy and anatomical integrity.
726 THE RESPIRATION AND [pt. m
4-15. Respiration of Mammalian Embryos
The data which we have on the subject of the respiration and heat-production of the mammahan embryo are very scanty and fragmentary. The question has been handled usefully from an obstetrical point of view by Harding; Murlin; and Feldman, but a great deal of the information contained in their reviews lies outside the scope of the present book, for it is concerned with changes in the maternal organs during pregnancy. The modern period was opened by Zweifel's discovery in 1876 that oxyhaemoglobin was to be found in the umbilical blood of an infant that had never entered on the pulmonary stage of respiration. This stimulated N. Zuntz to try some experiments in which he asphyxiated the pregnant animal. Most of the work was done by the simple method of ascertaining whether the blood in given blood-vessels was arterial or venous, light or dark, and in this way he found that on asphyxia the foetal circulation would give up oxygen to the placenta and so to the vessels of the uterine wall. Pfliiger himself added some remarks to Zuntz's paper, and the line of investigation was continued some years later by Cohnstein & Zuntz in collaboration. Their paper, which was very long, was concerned to a large extent with measurements of bloodvolume, enumeration of blood corpuscles, etc., which need only be mentioned briefly here. They were the first to discover that in the earher stages of development the number of red blood corpuscles in the foetal blood is very low, in certain cases only i or 2 being present for every 10 in the maternal blood. A curve constructed from the data of Cohnstein & Zuntz for the growth in the number of erythrocytes is shown in Fig. 440 (Section 17-1).
They also made a good many experiments on the blood-count of newly born infants, and gave in their paper all the literature on that subject before 1884. The early work of Quinquaud; Convert; Wiskemann; and Hoesslin had not succeeded in providing any data on the growth of the haemoglobin-content of the foetal blood, so Cohnstein & Zuntz turned their attention to that, and obtained some interesting results. Discussion of these, however, will be deferred to the section on pigments. Cohnstein & Zuntz also measured the bloodvolume in rabbit embryos, and their findings are graphically reproduced in Fig. 161. The volume (expressed as per cent, of the embryo) of the blood in the embryo rises; that of the blood in the placenta
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
727
and in the embryo plus placenta falls. Some figures for placenta weight in rabbits are included on the graph. Evidently the placenta circulation fills up with blood before the true embryonic circulation has had time to develop very far, and in the early stages the bloodvessels in the embryo form only a small part of the total placental circulation. All these facts have an obvious importance with relation to foetal respiration. Cohnstein & Zuntz in their paper gave a full bibliography of the earlier work on blood-volume in newly born infants and the young of animals. They also dealt with the changes in the blood following birth, the foetal blood-pressure, the foetal pulse frequency and circulation rate, using Ludwig's Stromuhr. They did not relate any of these determinations very closely with the age of the embryo.
Their most important work was done on the blood-gases of the embryo. They determined the oxygen and carbon dioxide content of the umbilical artery and vein, and found what change took place during an interval of 24 minutes. From the resulting differences they did not themselves calculate the respiratory quotient, saying that a much greater number of " Doppelanalysen " would be required, but this has often since been done from their figures (e.g. by Murhn) ; in one of their experiments it works out at i-6, in the other at 1-04. They calculated from their data the amount of oxygen used and carbon dioxide given out over a definite time per unit weight (metabolic rate), and obtained in the case of a sheep embryo of 1300 gm. the figure of i-i6 c.c. oxygen per kilo per minute. Comparing this with Reiset's figure for the adult sheep, of 5-8 c.c. oxygen per kilo per minute, they concluded that the embryonic metabolic
NEII 47
10 20 30 40
Weight in grams rabbit embyros Blood vol. Inyo fo In placenta +foetus of embryo s® " " only
weight (• In foetus only
Fig. 161.
728 THE RESPIRATION AND [pt. iii
rate was four times as small as that of the adult. The same conclusions applied to carbon dioxide.
These investigations led to a long-continued discussion in which a dichotomy of opinion soon presented itself Pfliiger, who had always affirmed that the metabolic rate of embry^os was far smaller than that of fully grown animals, welcomed Cohnstein & Zuntz's work as a confirmation of his views. The embryo, he said, has need for practically no muscular movement, and lives in a liquid of specific gravity very like itself, so there can be no necessity for great expenditure of energy, and therefore no " bemerkenswerthe Respiration". Gusserov took another view. Abstracting Pfluger's papers for the Archiv f. Gyndkologie in 1872 he said that, although it might be true that muscular motion was at a minimum in embryonic life, yet the astonishing rapidity of growth might equally well demand a considerable expenditure of energy. "You cannot overlook", he remarked, "the amazing speed with which the embryo passes from the tiniest size to the weight of the foetus at term, and this phenomenon can hardly take place without an active metabolism." Gusserov's words contain the origin of the notion of "Entwicklungsarbeit", afterwards so much elaborated by Tangl. But, although all those who took part in the controversy admitted that experiments alone could test the matter, none were carried out until 1900, when Bohr took it up anew. Cohnstein & Zuntz's second paper was only concerned with the arterial pressure before and after birth, the causes of foetal apnoea, and the first stimulus for pulmonary respiration at birth.
Bohr attacked the problem again with the advantage of improved methods, and he showed that the majority of the errors imperfectly guarded against by Cohnstein & Zuntz would act in the direction of making the metabolic rate too low. He abandoned the direct method used by them of estimating the blood-gases in the umbilical cord, and instead measured the oxygen consumption and carbon dioxide production of the maternal organism (guinea-pig) before, during, and after, clamping of the umbilical cord, i.e. cutting out altogether the influence of the embryo. In a typical experiment after compression of the umbilical cord the carbon dioxide excretion fell by 10 c.c, and the oxygen utilisation by 11 c.c, per 10 minutes. When the clamp was taken off the respiration at once rose to its former value, and fell again to just the same extent towards the end of the experiment when a ligature was put on the umbilical cord. In 10 minutes, therefore, the embryo gave out 10-5 c.c. carbon
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 729
dioxide and used up 11-5 c.c. oxygen; a respiratory quotient of 0-913. The following table gives the results obtained:
C.c. weight
Respiration per lo min.
(Carbon dioxide) metabou *■"
C.c. carbon
C.c.
oxygen taken in
Respiratory quotient
(c.c. per kilo
• per hour)
embryo
put out
Mother
Embryo
Embryo
Mother
35-8
IO-5
II-5
0-74
0-913
586
452
39-0
I2-0
100
079
I -20
462
408
6i-5
lO-O
9-0
092
i-ii
488
478
23-8
6-0
6-0
08 1
I -00
252
483
i6-o
50
30
0-91 0-87
1-67
1350
598
4-0
4-0
I -00
756
490
Average (excluding 5 gm. embryos) 509 462
A glance at the table shows that in all cases the respiratory quotient was in the neighbourhood of unity, from which it may perhaps be concluded, though Bohr himself refrained from emphasising it, that the main source of energy in mammalian development is carbohydrate^. During the periods when the maternal organism alone was respiring, the respiratory quotients varied between 0-74 and 0-92, with an average of 0-84, instead of the foetal average of 1-14. Perhaps most interesting of all are the figures for metabolic rate, calculated on the basis of the carbon dioxide results. There is a fairly close correlation as regards age, for the values run in order of embryo weight, 1350 (this was regarded by Bohr as doubtful), 756, 252, 586, 462 and 488. The third of these is the only aberrant one, and a first survey would conclude that the metabolic rate declines in the guinea-pig embryo from a very early time, just as it does in the chick. This point of view, however, does not fit in with the data of the calorimetric workers mentioned below, and, as a matter of fact, Bohr himself did not adopt it. He contented himself with averaging the figures and concluding that the metabolic rate in pre-natal life in the guinea-pig was of much the same order as in the maternal organism, thus agreeing with Gusserov rather than Pfliiger, and avoiding any commitment on the question of whether during development it went up or down. Bohr's position was, of course, that no true Entwicklungsarbeit was necessary, but that, at the same time, the embryonic cells were not "in a condition to exist without a vigorous metabolism". With his work all accurate information on the respiratory intensity and respiratory quotient of the mammaHan embryo ceases, and there is probably no gap in our
^ Again, Dickens & Simer found an R.Q,. of 1-04 for whole rat embryos in vitro (see p. 703).
47-2
730 THE RESPIRATION AND [pt. m
knowledge of the biophysics and biochemistry of the embryo at the present time so great as this.
Various investigations have been made of closely related subjects. The question of the initiation of pulmonary respiration, for instance, was thoroughly gone into by Preyer and by Bert. The passage of gases through the placenta was studied by Dubois & Regnard, by Butte and by Charpentier & Butte, who from clinical experience and a few experiments with rabbits supported Zuntz's original view that in maternal asphyxia the foetal can give up oxygen to the maternal circulation. In 1880 Hoeyghes reported that carbon monoxide would not pass the placenta, but this was shown to be incorrect by Grehant & Quinquaud and Plottier and, later, Nicloux and Nicloux & Balthazard, examined the passage of carbon monoxide across the placental membranes in the guinea-pig. They concluded that it passed through by a diffusion process provided that sufficient time could elapse for the slow dissociation of the carboxyhaemoglobin to create an adequate pressure gradient at the placental membrane. It then appears in the blood of the embryo, but, if the carbon monoxide is given in too great amount, death of the animal occurs before these events have had time to happen, and no carbon monoxide is to be found in the foetal blood. Huggett later investigated, in some careful experiments, the question of the passage of oxygen and carbon dioxide through the goat placenta, measuring the partial pressures in the maternal and foetal bloods with a view to deciding whether diffusion or secretion held good. The results were as follows:
Table 84. Embryonic blood-gases per cent, {average) .
Sheep (Cohnstein Goat (Huggett) & Zuntz)
Carbon Carbon
Oxygen dioxide Oxygen dioxide Foetal arterial blood from umbilical vein 796 29-9 6-3 40-5
Foetal mixed blood (going to brain) ... 5-90 36-6 — ~
Foetal venous blood from umbilical artery 2-94 41-4 2-3 47-0
The blood-gas tensions were as follows (in mm. of mercury) :
Goat (Huggett) f ^ \
Carbon Oxygen dioxide Foetal arterial blood from umbilical vein 41 44
Foetal mixed blood (going to brain) ... 28 54
Foetal venous blood from umbilical artery 15 61
Maternal arterial blood ... ... 60 43
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 731
The difference between the oxygen tension of the blood supplied to the embryonic circulation and the blood coming away from the embryo is therefore 60 — 15, i.e. 45 mm. of oxygen — amply sufficient to allow of a diffusion process^. In just the same way, the difference between the carbon dioxide of the maternal arterial blood and that of the blood coming away from the embryo is 61 — 43, i.e. 18 mm. carbon dioxide, and the same argument holds. The maternal and foetal bloods could not be compared directly in Barcroft differential manometers because they have not the same dissociation curve, so Huggett used a differential tonometer method in which gas mixtures were allowed to come into equilibrium with the maternal and foetal blood, and the tensions then estimated in a Haldane gas analysis apparatus. The relative blood-gas tensions then worked out as follows:
Maternal arterial blood (oxygen) _ i -g
Foetal venous blood (oxygen) i -o
Maternal arterial blood (carbon dioxide) _ 10 Foetal venous blood (carbon dioxide) i -2 1
Maternal venous blood (oxygen) _ i -o
Foetal arterial blood (oxygen) 1-3
"The first experiment shows", says Huggett, "that the gradients existing between the foetal venous blood going to the placenta and the maternal arterial blood going to the uterus are adequate for diffusion. The third experiment shows that the maternal venous blood in the uterine vein has a lower oxygen tension than the foetal arterial blood which is not surprising if we remember that the placenta, unlike the lung, absorbs an appreciable quantity of oxygen." Huggett also did some experiments in continuation of Zuntz's original ones, in which the foetal blood gave up oxygen to the maternal blood. By asphyxia Huggett found that this can actually take place. The reversal of the gas current through the placenta when the gradient on the maternal side is reversed, though difficult to explain on any secretion theory, would be an obvious corollary of diffusion.
^ Huggett worked with embryos at term, but Kellogg showed that the difference between O^-content of maternal arterial and foetal venous blood in the dog is much greater early in development than it is later on. The difference is gradually lessened by the rising oxygen-content of the foetal venous blood. This may be due to greater oxygen-carrying capacity or to an increase in the ratio placental area/unit foetal weight. Apparently the placenta gives a low margin of safety, for the pulmonary area of the newborn infant is at least twice the placental area, and the oxygen-content of its blood twice that of the foetus at term. Nor is the blood of the latter more than 63 per cent, saturated with oxygen, although the corresponding maternal figure is 95 per cent. (Eastman).
ell, etc.
Haselhorst
18-87
15-44 9-02 5-85
14-56
lO-II
3-53 0-87
732 THE RESPIRATION AND [pt. iii
Bell, Cunningham, Jowett, Millet & Brooks; and Haselhorst found the volume percentage of oxygen in the bloods to be as follows :
Maternal arterial ... Maternal venous ... Foetal arterial Foetal venous
Bell also made some observations on the alkaUne reserve of the two circulations, obtaining results contrary to the earlier ones of LevySolal, Weismann-Netter & Dalsace; Williamson; and Losee & Van Slyke. The English workers found more carbon dioxide combining power in the maternal than in the foetal blood, and the French and American workers found the opposite.
Table 85.
Carbon dioxide combining power in vol. % (c.c. per lOO c.c.)
Maternal Foetal Levy-Solal & associates (4 cases) ... ... 30-4 49-5
48-1 55-9
37-8 53-6
42-0 48-2
Art. Ven. Ait. Ven.
Bell & associates (5 cases), average ... 40-45 43*45 37'7 40"0
Losee & Van Slyke (4 cases), average ... 50-0 53-0
Williamson (7 cases), average 31-2 34-0
Rielander ... ... ... ... ... — 37'i
The role of the placenta in foetal respiration has also been studied by Schmidtt, who in a series of papers has put forward the suggestion that the foetal respiratory centre is situated, for a time at least, in the placenta, and not in the embryonic medulla. By perfusing the placenta with various solutions, and by studying the effect of altering the pH of the perfusing fluid, he found that the acid side would cause vaso-dilation and the alkaline side vaso-constriction, so that a regulatory mechanism of some sort was evidently present^.
4-16. Heat-production of Mammalian Embryos
We may now return to the heat-production of the mammalian foetus. Unfortunately this has never been measured directly, for the technical difficulties in doing so have so far been insuperable, and all that we know about it is derived from experiments in which the
^ There are, of course, no nerves in the mammalian placenta (Ikeda).
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 733
maternal organism enters in as a disturbing factor. Much attention has been given to the basal metabolism of women in pregnancy, and, although the conclusions about the embryo are not too certain, it is necessary to review them carefully.
In 1908 Rubner expressed the belief that his surface area law applied not only to the newly born animal but also to the embryo. As the average weight of an individual human infant at birth is 8 per cent, that of the mother, Rubner calculated that the metabolic rate of the foetus at term would be approximately twice the maternal metabolic rate, but, because the foetus is not very active, its rate would be less than that. Such a point of view agreed well enough with the experimental values of Bohr. But it was very difficult indeed to know what part the new tissues would play in the total heatproduction of the mother plus the foetus as a unit, for, although the embryo itself might have a much higher metabolic rate than the mother, the fluids, the umbilical cord, the membranes, etc., would have a very low one or none at all, while that of the placenta was more or less incalculable. The earlier observations on pregnant animals, moreover, gave conflicting results. Reprev could find no increase in basal metabolic rate in the pregnancy of the rabbit, guinea-pig, and dog, while, on the other hand, the figures of Oddi & VicarelH on mice showed a marked increase. This increase was also found by MagnusLevy, who carried out the first reliable observations on a pregnant woman; in this case the rate rose from 2-8 c.c. oxygen per kilo per minute in the 3rd month to 3-3 c.c. in the 9th month (17 per cent.). L. Zuntz however, did not find such a rise. Murlin in 1910 was able to show that the extra heat-production during pregnancy in the dog was almost exactly proportional to the number of embryos, i.e. the total weight of the litter. The experiment was done on two pregnancies of the same dog. The figures were as follows :
Gram calories produced per day During sexual rest (normal) ... ... 505'3
During first pregnancy ... ... 55 1 '3
During second pregnancy ... ... 763-8
In the first pregnancy i puppy was born, in the second 5. 55 1 "3 — 505-3 gave 46-0 cal. per puppy, or, as it weighed 280 gm., 16-4 gm. cal. per 100 gm. Similarly 763-8 — 505-3 gave 258-5 cal., or 51-7 cal. per puppy, or, as they weighed 3 1 2 gm. each, 1 6-8 gm. cal. per 100 gm. If now it could be shown that the metabolic rate of the maternal organism
734
THE RESPIRATION AND
[PT. Ill
^ ->
~
P
>. 640
_
/
^
/
l^aoo
y
/
^|580 9-^560
_
n
^
/
S ,40
I
U
O
i' 520
1
1
1
1
1
i
5
6
7
remained perfectly constant during pregnancy, there was a possibility of determining how the foetal metabolic rate varied. Murlin also estimated the heat-production throughout pregnancy, and a curve plotted from his figures is shown in Fig. 162. No appreciable increase occurred until gestation had been half accomplished. Murlin calculated that the extra metabolism due to the embryo (and all accessory structures) at the end of pregnancy was almost exactly equivalent to the amount which a newly born animal of the same weight would theoretically produce (according to Rubner's skin-area law) if exposed to ordinary room temperature and resting. Thus in the case of the I -puppy pregnancy, the extra metabolism was 46-0 cal., and the extra metabolism calculated by Meeh's formula from the embryo-weight was 45-4. Similarly the extra metabolism in the case of the 5-puppy pregnancy was 258-5, and the same calculated from the embryoweights was 251-6 gm. cal. Thus the total curve for mother and offspring should not suffer any change at birth, if all muscular movement were abolished. Murlin & Carpenter were later able to verify this in the case of man where there was no muscular movement.
The estimations of L. Zuntz; Murlin & Carpenter and Hasselbalch all agreed in showing an extra basal metabolism near term of about 4 per cent. Apart from this small increase the heat given off per unit weight per unit time, according to Murlin, was the same as under normal conditions, i.e. the embryo functions as so much maternal tissue, its higher metabolic rate being just counterbalanced by the inactive and relatively inactive structures. Another exact compensation was that the increase in oxidation of the infant's body when it passes from the warm environment of the uterus to the cold of the outside world was almost exactly equivalent to the oxidation rate of the accessory structures that supported it in utero.
Other researches on the basal metabolism during pregnancy in man are those of Baer; Cornell; Wilson & Bourne; Haselhorst & Plant; Root & Root ; Sandiford & Wheeler and Rowe, Alcott & Mortimer. The first two of these sets of data are believed by Harding to be faulty,
Weeks of pregnancy Fig. 162.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
735
but the others equate well with those of Magnus-Levy, and form the basis of our knowledge of the process. The gradual rise of heatproduction during pregnancy is particularly well shown in the figure of Root & Root, reproduced here as Fig. 163. From the 6th month it steadily rises until, at 6 weeks before birth, it is 23 per cent, higher than at 4 months. The total increase in weight, however, was only 14 per cent., and a non-pregnant woman showing a similar increase
Fig. 163.
in weight would only have increased her heat-production by 5 per cent., according to the tables of Harris & Benedict or Aub & Dubois. Similar results were obtained by Sandiford & Wheeler. Murlin & Carpenter had shown in 1 9 1 1 that if the energy exchange of a pregnant woman at the gth month were compared with the energy exchange of the mother post partum, the metabolism total in each case was exactly the same except for a balance of 4 per cent, in favour of the pregnant woman. This means that there is no deflection in the energy consumption curve at birth, that the maternal organism and the foetus function as two separate units in their consumption of energy, arid that the rise of heat production during
736 THE RESPIRATION AND [pt. iii
pregnancy is entirely due to the embryo. This attitude is adopted by Garipuy & Sendrail as the result of their work^. Sandiford & Wheeler have made this point of view very much the most satisfactory by calculating from their own figures and those of all other observers the (basal) metabolic rate of the embryo on this assumption. The weight of the foetus was obtained by means of the standard curves for human pre-natal growth, and Lissauer's formula for infants (10-3 x\W^ instead of Meeh's 12-3 x\/W^) was used to calculate its surface at the different stages. Then the weight and surface (Dubois charts) of the pregnant woman being known, and the weight of the foetus subtracted from it, the metabolic rate of the mother alone could be calculated. The result was that the sum of the two agreed remarkably well with the figures actually found experimentally.
More explicitly what Sandiford & Wheeler did was this. Sandiford had previously calculated the surface area of the human embryo at different stages, and this value, added to the calculated surface area of a woman equal in weight to the pregnant woman minus the foetus, gave the total surface in question. Then, when the total calories eliminated by mother and foetus were divided by the sum of the surface areas so obtained, the resulting figures would represent the heat production of a unit mass of active protoplasmic tissue. It was found that actually there was no significant change during pregnancy in this value, which remained constant within small limits of variation at 35 calories per square metre per hour. Sandiford and Wheeler pointed out that this calculation would be invalid if surface area law depended on Newton's Law of Cooling, but was valid if it depended, as in their opinion and in that of Boothby & Sandiford, it did, on a proportionality between surface area and mass of active protoplasmic tissue. I shall again return to this point. They also used another method of calculation. They assumed that the heat-production per unit mass of foetal tissue was constant throughout foetal life, and, by multiplying the heat-production for each square metre of body-surface each hour by the surface area of the foetus corresponding to its estimated weight, the total calories each hour for the foetus were obtained for the various months. If this latter figure was subtracted from the total calories of mother and foetus, the total calories of the mother alone were obtained,
^ See also Pommrenke, Haney & Meek.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
737
and when divided by the estimated surface area of the mother, the calories per square metre of body-surface for mother alone were practically the same as those found by the other method. But they were not quite the same, and the difference between them was always greater towards the end of pregnancy than at the beginning — a fact for which Sandiford & Wheeler offered no explanation, but which may be due to the fact that the heat-production per unit 72
time per unit mass of foetal tissue is probably not the same throughout development. The general trend of all that has already been said works strongly against the assumption that it is. The various curves which have been mentioned are shown in Fig. 1 64 taken from Sandiford & Wheeler's paper. Curve A represents the calories for each kilo (in this instance not rising very much) , curve 5 the basal metabolic cd -io rate calculated during the -2 0.94 course of pregnancy by t ^_^^ dividing the total calories put out each hour by the sum of the surface areas of mother and foetus, and comparing the result obtained
with the Dubois normal of the mother, 36-5 calories. Curve B' shows the basal metabolic rate calculated in the usual way, using the Dubois surface area and the normal standards. Curve C shows the calories per square metre per hour calculated in the same way as for curve B, and curve C shows the calories per square metre per hour obtained by dividing the total calories each hour by the Dubois surface area obtained by using the total weight of mother and foetus in the usual manner. Curve D represents the total calories per hour eliminated, i.e. the raw data of heat given up to the calorimeter.
.5? 1.68
og ro-e64 " 1. ■5g.60
f2 56
52
I1.38 s_ o
+ 10
0-78
, 1
1
5 Z
J
I -JU U
70 V
) ^ 4
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r^---^^ -^^^^fe
) ^^^C^B^^^ -.^
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X ^z \
^^r^^ k ^L ^^'
m ^--^
^ 6 8 10 2 4 Lunar months
Fig. 164.
738
THE RESPIRATION AND
[pT. m
The general impression left by the work of Sandiford & Wheeler and of the other investigators mentioned is that the metabolic rate of the human embryo does not change much during pre-natal life.
3*30
3.00
2.70
2'40
2»10
1.80
Calories
1»50
1.20
•90
A
•60
V
•30
a
= Magnus -Levy
= Benedict and Collaborators
= Murlin 5? »?
= Palmer -Means -Gamble
= Sage Institute
L ! \
10 Years 15
Fig. 165.
20
28
But it must be remembered that they could not take account of the embryo until the 4th month, owing to its minute weight compared to that of the mother. On the whole, however, the measurements of heat-production during pregnancy are very difficult to interpret from the point of view of the present discussion.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 739
After birth, the metabohc rate is not a constant, nor does it begin to decHne immediately. In man it rises until about the 4th year, after which it declines continuously except for a slight kink at 12-5 years, believed to be associated with puberty. The classical paper in which this peak was observed is that of Dubois, which appeared in 1 91 6, but exactly the same state of affairs is indicated in the papers of Benedict & Talbot; Murlin & Hoobler; Murlin & Bailey; and Marine, Lowe & Cipra. Dubois himself made many measurements of the metabolic rate of children from 6 to 14 years of age, and compared them with those of many other observers for the periods before and after these limits. These papers should be consulted for the relevant literature. In Fig. 165, taken from his paper, the resulting curve is shown, and, as has already been said a definite peak at 4 years of age is to be seen. This curve is in every way comparable with that in Fig. 133, where the data of Gayda on the metabolic rate of the toad are plotted. The figures of Magnus-Levy & Falk, moreover, show the same peak on the basis of carbon dioxide and oxygen determinations. Clearly if this peak were real, it would be expected that infants born some time before term would show a basal metabolism below that of normal infants. This was actually found to be the case by Marsh; Murlin & Marsh; Talbot & Sisson and by Talbot, Sisson, Moriarty and Dalyrymple, whose figures agree very well and demonstrate a little further the earliest part of the Dubois curve.
Just as in the case of the toad the peak occurs not very long in the life-span after hatching, so in the case of the human being it occurs not very long after birth. But these two organisms are not the only ones for which a peak of metabolic rate has been demonstrated, for Deighton & Wood in 1926, using Capstick's calorimeter, found an exactly similar one in the case of the pig. Fig. 1 66 a shows the results obtained. From birth the pig's metabolic rate rises rapidly, reaching a maximum of 72 calories per square metre per hour at an age of 4 months, after which time it falls away less rapidly than it rose, and this peak holds true also when the heat-production is plotted against the weight. Not only the nature of the curve but also even its general form are in close agreement with the work of Dubois and many others on man, and Gayda on the toad. But Wood and his assistants found that not all breeds of pig gave a peaked curve. If the Berkshire breed was used instead of the Large White, the falling part of the curve appeared but not the ascending part, as is
740
THE RESPIRATION AND
[PT. Ill
indicated on Fig. 1 66 a by the small crosses, so that in this other breed the peak, if there was one, occurred before birth. That there had been one Wood had no doubt, for if the curve had been simply continued in an upward direction with decreasing age, the embryo of a few grams would have been radiating heat to the intensity of a red-hot body. The descending metabolic rate curves must, indeed, in all cases, come from a peak, and not from some indefinitely high level.
80
70
a 60
s
a K. 50
40
30
Subsequent work by Deighton on other breeds of pigs has carried further the work reported in Wood's paper, and has demonstrated that in some cases the peak occurs after birth, in other cases, before it. Russell, who hoped to find an extra-uterine peak in the rabbit, which is born relatively early, and an intra-uterine peak in the guinea-pig, which is born relatively late, found a curve for the former animal which resembled that of the Berkshire pig. Ginglinger & Kayser, however, did succeed in finding such a post-natal peak in the case of the rabbit, as may be seen from Fig. i66 ^. They contrast the curves given by the pigeon (showing neither chemical nor physical heat-regulation at birth) with the rabbit (which shows chemical regu
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o
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/ X ^^^^. ^ ^^~^ ^^^:» X " ^ - .^^ _
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10 12 1 66 a.
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- Man
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O Large white
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
741
lation only) and with the guinea-pig (which can fully control its temperature at birth). Correspondingly the guinea-pig — like the Berkshire pig — obeys the classical rule of descending heat-production from birth onwards ; these animals have, as it were, settled on their thermal neutrality point before birth and as it is kept steady we can observe the effects of decreasing relative surface. The pigeon and the mouse, on the other hand, are born without any regulatory power, and their metabolic rate goes on increasing until this is attained. Intermediate between these two groups come such animals as the rabbit, which can
A
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\
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1
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X N
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/ ^
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-^
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-Pin
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3 5 7 9 11 13 15 17 19 Mulblples of the initial weight Fig. 166 ^.
resist cold at birth but not heat, i.e. which have a partial regulatory power. These show an increasing metabolic rate for a short time only, and the curve for the rabbit in Ginglinger & Kayser's figure ( 1 66 b) compares interestingly with that for man in the graph of Dubois ( 1 65) ^. And the fact that the chick's metabolic rate is declining throughout its incubation-period from the 5th day onwards would agree with the finding that its heat-regulative power is fully developed at birth. The peak of metabolic rate in mammals is explained by Ginglinger & Kayser, then, as being due to their varying degrees of heat-regulative power, but would this explain the peak on Gayda's curve for the toad, which never succeeds in regulating its temperature at all ?
1 And with those for growing calves given by Brody.
742
THE RESPIRATION AND
[PT. Ill
4-17. Anaerobiosis in Embryonic Life
Very few observations have been made of the respiration of embryo cells in tissue culture, but Burrows has some interesting experiments in this direction. Taking explants of chick embryonic heart cells, he placed them in different partial pressures of oxygen, and found that their behaviour was quite different according to the stage of development of the chick from which they had been taken. The results he obtained are pictured in Fig. 167, from which it can be seen that fibroblasts from 4-5-day embryos would grow and pulsate for 46 hours or so in ^^ pure nitrogen, and for 50 hours in only 7-5 per cent, of oxygen. Fibroblasts from i o- 1 5-day em- 5 30 bryos, however, would not grow 8 at all in nitrogen, and only for -^ 12 hours in 1-5 per cent, oxy- °2o gen. Burrows was in doubt as to the meaning of these phenomena, but explained them by 10 the hypothesis that there must be some source of energy contained in the young cells, which the older ones have not got. One is reminded, on the one hand, of the work of Cohn & Murray on the growth-rate of embryo explants, and, on the other hand, of the concept of "ontogenetic momentum" which Byerly's results on asphyxiated embryos (see p. 607) and de Bruyne's results on embryo autolysis (see Section 14-11) have brought into being. Consideration of it will be postponed till later in the book. Burrows found, in a word, that with age the property of being able to grow anaerobically declined, and was eventually lost. This important result was fully confirmed by Wind, who modified it by using really strict anaerobic conditions. Under these only the very slightest amount of growth went on even with heart-cells from 4-5day old embryos, but when an atmosphere of 2-10-* vol. per cent.
10 15
Days of development
Fig. 167.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 743
of oxygen was used, the difference was unmistakable, for cells from lo-day old embryos would not grow at all, while those from 5-day old embryos grew excellently. Wind also made the very interesting observation that, if the cells were in each case sub-cultured for many weeks and then brought under anaerobic conditions, the difference between the cells from lo-day embryos and those from 5-day embryos had entirely disappeared. No difference in growth was now perceptible.
Work along these lines was continued by Wright whose results may be summarised in the following table :
Table 86.
Mm. mercury
pressure of
oxygen
Wright Explants of loth day chick embryo myoblasts:
(a) In 0-22 % glucose : mitosis ceases at
emigration ceases at
(b) In o-o6 % glucose: mitosis ceases at
emigration ceases at ,, Jensen rat sarcoma: mitosis ceases at
,, Mouse carcinoma: mitosis ceases at
Ephrussi, Chevillard, Explants of 8th day chick embryo heart fibroblasts :
Mayer & Plantefol mitosis (but not movement) ceases at ... ... y-o
These facts lead naturally to the consideration of the evidence which has been brought forward from time to time in favour of the view that during the early stages of embryonic development anaerobiosis may be possible, or may even normally occur. There is no need to dwell on the first efforts of the workers on the hen's egg, for their experiments have already been briefly described. Nor can cleavage of echinoderm eggs go on in the absence of oxygen. Some recent careful experiments of Amberson on the eggs of Arbacia punctulata have demonstrated that cleavage proceeds at normal rate down to very low oxygen tensions — about 1 1 mm. of mercury, between which point and 4 mm. the rate is slowed down, while below 4 mm. cell-division in this egg will not go on at all. Drastich finds exactly parallel effects in the case of Strongylocentrotus lividus. In the case of the nematodes, again, Zavadovski showed in 19 16 that cleavage in Ascaris megalocephala would not go on in the absence of oxygen and that the reason why it stopped in putrefying media was because the bacteria were successfully operating a prior claim for the oxygen present. More recently, Zavadovski & Orlov have demonstrated the absolute dependence of many nematode embryos on oxygen for their cleavage,
744 THE RESPIRATION AND [pt. iii
and Kozmina, working on Ascaris, has obtained very^ similar results to those of Drastich, mentioned above. On the other hand, the formation of the chitinous envelope, the internal membrane and the perivitelline space, and the elimination of the two polar bodies, can go on normally in the absence of oxygen, as appears from the work of Szwejkovska. As regards cleavage, Szwejkovska is in agreement with Kozmina and with Zavadovski & Orlov,
There is little evidence that developmental rate in any form is increased by raising the oxygen tension or concentration. Rollat's claim that silkworm eggs hatched much earlier in compressed than in ordinary air was discredited by Bellati & Quajat.
The disputed question only concerns amphibia and birds, and originates from the work which Samassa did during the last decade of the last century. We are not here concerned with variations in the degree of susceptibility to oxygen lack or oxygen excess on the part of the embryo during its development, but with the capacity which it has been alleged to have of being able to live and develop anaerobically in the very early stages. Samassa affirmed in his first paper that the early segmentation stages of frog's eggs were apparently independent of oxygen, and would proceed in atmospheres of hydrogen and nitrogen, though gastrulation would not take place under such conditions. Kept in pure irrespirable gases, they retained, if unfertilised, their developmental capacity for many days, though the resulting embryos were often abnormal. The experiments were done in a stream of pure hydrogen, so that Samassa believed he had washed every trace of oxygen out of the gelatinous egg-coverings. He next tried high vacua, first by means of a mercury pump, and then a cathode ray. Still they developed as far as complete blastulae. Samassa found, however, that carbon dioxide had a definitely toxic action, stopping segmentation, and, if pure, killing the eggs within 20 hours. (Compare Burfield's work on the plaice egg, p. 669.) He recalled that Hallez had found Ascaris eggs to be capable of living for a month in pure carbon dioxide and then developing, but rightly regarded a parasitic nematode as a special case. About the same time Loeb reported an extreme resistance on the part of Fundulus eggs to oxygen lack in the very early stages. Samassa was convinced that the effects he obtained were not due to traces of oxygen, and argued that, if it were so, all the eggs in a vacuum flask would hardly be expected to develop synchronously, for the weakest would be
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 745
weeded out, yet this was never the case. Again, a high population of eggs would be very injurious, yet this was not so. Then it might be supposed, if traces of oxygen were responsible, that admission of air and re-evacuation would lead to further development, but this was not the case. In atmospheres of 20 per cent, oxygen and 80 per cent, hydrogen, the eggs developed just as normally as in the pure hydrogen. Loeb supported Samassa's conclusions, working with Ctenolabrus and Arbacia eggs. In atmospheres of hydrogen, he found, development would stop, but by no means immediately; thus the eggs of Fundulus (a bottom fish) could last 15 hours in complete absence of oxygen, segmenting, and retaining perfect viability even after 4 days' anaerobiosis. The eggs oi Ctenolabrus, on the other hand, were very sensitive to carbon dioxide, and a stay of only 4 hours in that gas killed them altogether. Arbacia and Paracentrotus eggs were held up at once in the absence of oxygen, a fact subsequently confirmed by Warburg. Again, in later stages, the hearts of Ctenolabrus and Fundulus behaved rather differently. In 10 mm. partial pressure of oxygen the heart-beat of Ctenolabrus embryos was quite abolished, but that of Fundulus embryos could proceed, if slowly, for as long as 9 hours, and then completely recover. Carbon dioxide was equally toxic for both hearts, and hydrogen would act as an antidote to its action so that the heart might begin beating again in hydrogen when that gas was substituted for carbon dioxide.
O, Schultze opposed these conclusions. He maintained that the technique of the other workers had been faulty, and that traces of oxygen had been present. As a means of removing all such traces from the air, he adopted the ingenious expedient of using the eggs themselves. The eggs were placed in a tube (of just the right size to fit them) passing through a cork. After 2 days, eggs Nos. i and 8 (those nearest the open ends of the tube) were gastrulating, 2 and 7 were beginning to do so, while Nos. 3, 4, 5 and 6 had all stopped in the earliest cleavage stages. When all were turned out into a dish, however, all developed normally. Schultze concluded that small amounts of air must have been present in the other experiments, but it is as a matter of fact very difficult to see from Samassa's account of them how this can have been so. As for Loeb's results, Schultze interpreted them as being simply due to a difference in oxygen requirement as between the two embryos. Nevertheless, investigators continued to report confirmations of Samassa's experiments, notably Godlevski in 1901, and contra 48-2
746 THE RESPIRATION AND [pt. iii
dictions of them, such as Wesselkin in 191 3, who worked with chick embryos, and found that an atmosphere of 5 per cent, oxygen killed them in 48 hours, but that 10-15 per cent, permitted continued development for 72 hours. It may be concluded that, although there is no strong reason for believing that embryonic development can ever go on anaerobically, there are yet some curious facts which ought to be looked into before a final decision is reached. And the work of Reiss & Vellinger referred to on p. 869 suggests that the energy required for cleavage may be obtainable without free oxygen, by electron transfer.
4-18. Metabolic Rate in Embryonic Life
Attention must now be directed for the last time to the metabolic rate question. We have already seen that Gayda (for the toad). Wood (for the pig) , Dubois (for the human being) and Ginglinger & Kayser (for the pigeon and the rabbit) have shown that the respiratory rate has a point of maximum intensity during the life-span, though subsidiary kinks on the curve may exist (as at puberty). It is extremely probable that, in the rising rate curves of echinoderms and amphibia early in development, we see the ascending part of a curve, and, in the falling rates of avian embryos, we see the descending part of the same curve. What factors determine the point in the life of the individual at which the peak shall occur are as yet obscure although, as we have seen, Ginglinger & Kayser explain it by the onset of heat-regulation .
It must first be pointed out that the falling part of the curve has often been plotted, and is well seen in the measurements of MagnusLevy and Falk on man ; of L. Mayer on the hen, the duck, and the guinea-pig ; of Sayle on dragonfly larvae ; of Krarup on rabbits and of Benedict & Riddle on pigeons^. Mayer noticed that, in the case of the guinea-pig, the fall after birth followed the course of a regular hyperbola. The following figures, again, exemplify it.
C.c. carbon dioxide put out per kilo per hour Very small rabbit embryo (Bohr) 750
Rabbit embryo nearly at term (Bohr) 509
Adult rabbit (Krarup) ... ... 450
Its extremely general character is shown by the fact that Hee obtained a curve for intensity of gaseous exchange during the develop ^ Other instances are cows (Brody) ; cladocerans (Obreshkove) ; molluscs (Hopkins) .
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 747
ment of the mould Sterigmatocystis nigra which closely resembled that of a growing population of cells such as the chick embryo. Among the most important of the suggestions that have been made with regard to it is the notion of the "masse protoplasmatique active", in contrast to the "ballast", which was introduced by Friedenthal, and has since been much developed by the Strasburg school (Faculty of Medicine). As differentiation goes on, and the make-up of the embryo becomes ever more and more complicated, there must be a constant increase in the amount of storage substances which have themselves no respiratory function, but which participate in the total weight of the embryo. These substances, which are known as "substances paraplasmatiques", will, if they increase out of proportion to the size of the growing embryo, obviously have the effect of lowering its unit respiratory activity. Cohn & Murray suggest that the lipoid granules of the central nervous system and the brown pigment of the heart and liver may be substances of this class. Kassowitz and Miihlmann have discussed the accumulation of "metaplastic" bodies in cells during the growing process.
Another and most important factor which has to be taken into consideration in this connection is the relation between the surface and the volume. As Cohn & Murray put it, " It is in the very nature of geometric relations that with growth the \'olume or mass increases as the cube, and the surface as the square of a number. The result from a biological standpoint is that for a unit mass of active protoplasm undergoing continuous chemical changes, the portals, that is to say, the surfaces of the organism for entry and exit of the substances which are the antecedents or products of vital activity become continuously smaller, and therefore continuously less suitable for maintaining the original velocity of metabolism. There must necessarily follow a diminution of activity and all the other changes that are merely the logical outcome of the initial modification."
The surface/volume theory must, however, be used with care, for it has various implications other than those which appear at first sight. If the embryo maintained a perfectly spherical shape as it grew, then the theory could be applied to it in its simplest form, but the active surfaces are so numerous and so large that a great number of complicating factors must, at any rate, be admitted into the discussion. Thus in the case of the chick embryo, its effective surface is not only its skin, but also a collection of structures such as the
748 THE RESPIRATION AND [pt. iii
membranes of the allantoic and amniotic sacs, the blastoderm covering the vitelline membrane, and the renal tubules and glomeruli. As the chick is a metazoan animal, there are also the individual surfaces of the cells to be considered. Murray has summarised as follows a number of the points which have to be borne in mind in considering this question, "(i) In the case of individual cells of metazoa which are, as far as we know, of about the same size throughout life, the average of their surface/volume ratios would not change with development any more than it would in a growing colony of unicellular organisms. (2) The surface/volume ratio may theoretically be maintained at any level simply by the infolding or wrinkling of the surface, as is seen in the intestines. (3) In actuality the area of capillary surface is adjustable since the development of new vessels such as is seen in the processes of repair may occur as the result of the repeated vigorous functioning of a part. (4) It is known that under normal conditions only a fraction of the capillaries and therefore of an exposed surface is open or active at any one time. For instance the amount of heat-radiation from the skin actually depends less upon the measured skin surface than upon vascular changes, which, in turn, depend upon the metabohc rate rather than vice versa. (5) It is not only the area of the surface but the permeability of a surface that is important, and as the chemical constitution of each cell changes markedly with age, so will the surface permeability change. (6) The hypothesis that growth is correlated with the area of absorptive surface supposes that through a given unit of surface a certain restricted number of molecules may pass in unit time. But if, as we know, there is change with age in the kind and therefore the size and migration-rate of molecules which enter the cell, one would hardly expect this simple relationship to be maintained. (7) Growth or storage is the difference between absorption and elimination. Either one of these factors may vary more or less independently of the other and thus growth is necessarily dependent on both of them. As the ratio of storage to elimination changes with age, if absorption is dependent upon surface, growth cannot be, and vice versa It is nevertheless much to be wished that accurate measurements were available of the extent of the active surfaces in the chick embryo at all stages of its development.
The whole question is, of course, bound up with the controversy on the relation between heat-production and heat dispersal, " thermo
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 749
genese" and "thermolyse", a controversy now almost a century old and still vigorously proceeding. The two points on which all the disputants are agreed are ( i ) that animals give off to the calorimeter more calories per kilogram the smaller they are, and (2) that, per square metre of skin surface, they all give off much the same amount of heat. These two generalisations apply certainly to homoiotherms, and probably also to poikilotherms, with certain reservations. But the great divergence of opinion arises when we try to decide whether the surface area is the cause or the effect of the heat loss. Two schools of thought have come into being on the question. For one school the essence of the interpretation is Newton's law of cooling; a given amount pf surface necessitates inexorably a certain loss of heat, which must consequently be supplied by the protoplasm of the body; "thermolyse" is the cause of "thermogenese". The factors which cause this intenser metabolism in the case of the smaller creatures, i.e. those which have most surface in proportion to their weight, are, for the adherents of this view, all of one kind, and involve differences only of degree. These differences are regarded as being due to anatomical factors. "The tissues of the various homoiotherms — very similar in composition", says Terroine, "have a more or a less intense metabolism because by the perfectly coherent operation of the circulatory and respiratory apparatus, they receive in unit time variable amounts of food and of oxygen." Obviously on this view all protoplasms are identical, and the protoplasm of the tgg of a mouse could equally well go to form an elephant if it were not for the fact that the eventual form, shape, size and anatomical arrangement of the mouse exists in potentia in the egg-cell of the mouse, ensuring that the end-product of development shall be an object like a mouse with a proportionately large surface. The mouse protoplasm is, as it were, exactly the same as the elephant protoplasm, but destined to work a great deal harder because something in the mouse ^gg arranges that development shall stop when a certain small size is reached, and therefore that the energy turnover in unit time shall be considerable. The eventual surface and the eventual morphology, on this view, are what is originally given in the egg-cell — an almost Aristotelian conception which suspiciously resembles the proposition "au commencement etait la forme". Perhaps we may see in this attitude another expression of that point of view which has been so ably discussed by E. S. Russell in his book Form and Function.
750 THE RESPIRATION AND [pt. iii
Those who have not accepted these opinions have chosen rather to agree with St Paul's affirmation that "all flesh is not the same flesh, but there is one kind of flesh of men, another flesh of beasts, another of fishes, another of birds", and to maintain that protoplasms are not identical. A mouse diflfers from an elephant not because the surface was the element given in the first instance, and heat must be provided to compensate for that escaping at the surface, but because the metabolic constitution of its protoplasm differs, and therefore its surface. Or, in other words, the circulatory and respiratory systems do not govern the respiratory intensity of the tissues, but on the contrary were themselves laid out to meet a certain demand. "Thermogenese", in other words, is for these thinkers the cause of "thermolyse". An animal grows until it reaches a point at which its surface cannot be further reduced proportionately to its weight without involving a failure to carry away the appropriate portion of heat generated. An animal has, or may have, a surface proportional to its heat-production, and not a heat-production proportional to its surface. The fact that the heat loss per unit surface is much the same in most animals is regarded as a coincidence due to a curiously exact concordance between surface and active protoplasmic mass, due perhaps to the fact that both of them have a regular relation with the weight in normal cases. As for the egg, it does not contain the potential surface of the animal, save indirectly, for it consists of protoplasm capable of a definite metabolic intensity, or rather of following a definite curve till it arrives at a definite metabolic intensity, and upon that eventual intensity the eventual surface of the organism will depend.
The names associated with the first of these two points of view are numerous. Von Bergmann, one of the earliest workers on basal metabolism, advocated it, and in the earlier papers of Rubner it was fully adopted. In France, Richet consistently made it the basis of his opinions on these problems, and recently it has found a very vigorous defender in Terroine. The second of the two points of view was that originally held by Sarrus & Rameaux in 1838, and subsequently by von Hoesslin; Krogh; Putter; and Pfaundler. Its principal representatives recently have been the American school (Benedict; Lusk; and Boothby & Sandiford), and certain continental workers, such as Noyons; LeBreton; Schaeffer; and Kayser. It is certainly not possible yet to decide which of the two great groups of investigators
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 751
is in the right. But the well-known experiment of placing a homoiothermic animal at thermal neutrality so that there is no inducement for it to give out heat, with the result that it still does (Rubner and Terroine & Trautman), is not favourable to the Newton cooling law. Benedict again, working with abnormal weight/surface relations in man, such as occur in athletes or very obese men, in atrophic children or in men without limbs, found greater variations from the surface law than could be accounted for as experimental error. Benedict; Pfaundler; and LeBreton were all led to speak of an active protoplasmic mass, with which the actual surface might or might not be in exact direct proportional relation. Terroine, while accepting the notion of active protoplasmic mass to a certain extent, held that it was just that factor which had been adapted to the surface, and so remained firm in his conviction that the surface was all along the dominant factor. One fact, indeed, seems to have remained quite unmentioned in the various discussions which have taken place on these subjects, namely, the rising metabolic rate of echinoderm, molluscan and amphibian embryos. There the surface is moment by moment getting smaller and smaller relatively to the increasing weight of respiring protoplasm, and yet the metabolic rate, whether expressed as gram calories produced per gram per hour, or oxygen taken in or carbon dioxide eliminated per gram per hour, is steadily rising. If the surface were always the responsible factor this could not be taking place. It was indeed always a little difficult to understand what the adherents of the first of the two views (namely that "thermolyse" is the cause of "thermogenese") imagined to take place during embryonic development, for in the early stages the surface would be far greater in proportion to the weight than at any other time during life, and the egg-cell would be hard put to it to satisfy the heat-dispersing demands of its surface. Again, as Deighton and many others have pointed out, if the metabolic rate of the higher animals fell during embryonic development at the same velocity as afterwards during post-natal life, the single egg-cell must have been practically red-hot. That a peak on the curve must exist was overwhelmingly likely a priori, and, as regards the toad, the pig, and man, it has been actually found, but the existence of such a peak can hardly be allowed for on the von Bergmann-Rubner-Richet-Terroine theory, for, as far as we know, it is not associated with any considerable changes of surface area, and on their views it would have to be. It
752 THE RESPIRATION AND [pt. iir
must be admitted that the probabilities are much in favour of LeBreton's interpretation, as far as the embryological evidence is concerned. In post-natal life, as we have seen, it has often been assumed that the surface is somehow proportional to the active mass, but in the embr^'o this may not be so. The individual organism, as regards its protoplasmic metabolic intensity, follows the curve of its species, and comes at last to an equilibrium point, at which just enough surface has been developed to carry away conveniently the heat arising from its hereditarily determined protoplasmic metabolic intensity.
The surface is not the only entity that grows more slowly than the total body-weight. Brody showed that the instantaneous percentage growth-rate of oxygen utilisation and carbon dioxide elimination in the chick embryo is not the same as that of the whole body^. This affords an obvious indication of an active protoplasmic mass growing (like the surface) less rapidly than the embryonic body as a whole. If Brody's instantaneous growth-constants are compared they work
out as follows :
Table 87.
Carbon dioxide production Days Atwood & Weakley Murray Hasselbalch
Time taken to double the entity
Instantaneous % rates
growth
0-4 • 0-7
4-14
2-2
1-9
2-2
0-4 98
4-14 32
Growth in wet weight Days Lamson & Edmond
4-8
1-2
8-12 1-9
12-16 2-9
4-8 56
8-12 36
12-16 24
Days
Hasselbalch
Murray
6-10 1-2
i'5
10-14
2-4
2-1
14-18
3-6 31
6-10 56
47
10-14 29 33
14-18
19 21
"Either the COa-producing mechanism", says Brody, "develops at a constant percentage rate independent of the increase in bodyweight, or the weight of the body or its constituents cannot be taken as an index of the growth of metabolising tissues." These important facts illustrate from an unusual angle the constancy of heat output per unit surface. The surface is not the governing factor, but rather something else growing also more slowly than the total body-weight. There is no need to discuss the attempts which have been made to identify chemically this active protoplasmic mass, or the paraplasmatic proteins, for they do not directly concern the embryo.
1 See Figures 526 and 536.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 753
They may be found discussed in the books of Terroine & Zunz and of Lipschiitz. LeBreton & Schaeffer, however, in their work on the nucleoproteins of the chick embryo, were led to regard the paraplasmatic substances as mainly proteins from the fact that the nucleoplasmic ratio, chemically determined, fell with age in the chick embryo. Other proteins were therefore rising, and the active mass, they thought, could be represented by (though of course it was not regarded as identical with) the nucleic acid nitrogen as percentage of the total nitrogen. Some typical figures for the chick embryo were:
Chemical nucleoplasmic ratio Days of (Nucleoprotein nitrogen x ioo)/(Total
development nitrogen - nucleoprotein nitrogen)
8 IO-7
6-65
4-9
35
and naturally LeBreton & Schaeffer emphasised the similarity between the fall in nucleoplasmic ratio and the fall in metabolic rate. Cahn, working under their influence, found in a study of atrophy in muscle during starvation that the nucleoplasmic ratio changed. Normal muscle contained for 100 gm. of total protein 0-684 gn^' of nucleic acid, but atrophied muscle 0-961 gm. of nucleic acid for every 100 gm. of total protein. This was regarded as support for the theory of paraplasmatic substances, but the interpretation has been much criticised by Terroine. Terroine & Ritter carried out experiments which were the converse of those of LeBreton and Schaeffer on nucleic acid, for, instead of taking the same organism at different ages (and therefore sizes), they took different organisms of various sizes. Their results worked out thus :
Purine nitrogen in grams per 100 gm. wet weight of tissue
(
Muscle
Liver
Ox
o-o6i
0-146
Horse
0-079
0-125
Pig
0-074
—
Sheep ...
0-077
0-135
Dog
0-062
0-155
Rabbit
0-078
0-148
Hen
0-071
0-150
Pigeon
0-108
0-147
Rat
0-076
0-160
They concluded that these animals contained almost exactly the same amounts of nucleic acid in their cells, and that there was
754
THE RESPIRATION AND
[PT. Ill
no relation between heat-production and this factor, but as they neglected to estimate the total nitrogen, and so did not calculate the chemical nucleoplasmic ratio, their figures cannot be compared directly with those of LeBreton & Schaeffer for change with age.
Then Moulton's figures on the cow for nitrogen content of whole animals have often been taken
'^ uiciprifctyrri
Liver
10
S 5
Diaphragm Muscle
as evidence in favour of a direct relation between surface and total protein, but for small animals, Terroine, Brenckmann & Feuerbach could not find such a condition. Terroine, in his review of the subject, considers various other possible measures of the active mass, such as the work of Mayer & Schaeffer on lipoids, and that of Lapicque & Petetin on mineral constituents. He is, of course, concerned throughout to show that, as far as the evidence goes, the composition of all animals is practically the same, and cites with special approval the investigations of Abderhalden, Gigon & Strauss, of Osborne & Jones and of Osborne & Heyl, who found very similar amino-acid distributions in proteins from a mammal, a bird, a fish and a mollusc. Nevertheless, such arguments are not at all convincing, for they essentially consist in minimising the differences which have been found to exist, and it would be equally justifiable in the present state of our knowledge to emphasise small variations just as much. The fact is that we cannot as yet be sure on the ground of the chemical analyses we have whether protoplasms of different animals are different or the same, and, until a great deal more work has been done,
Q. I. O
Fig. i(
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 755
speculation will remain unprofitable. At the same time, it is quite legitimate to adopt an interim belief, either with Rubner; Richet; and Terroine, that they are the same, or with Pfaundler; Benedict; and LeBreton, that they are different. Certainly all the evidence from chemical embryology supports the latter view.
4-19. Respiratory Intensity of Embryonic Cells in vitro
Obviously the only way to decide whether surface is the governing factor in heat-production is to remove the surface, and to see whether the heat-production is then the same. This can be done by experiments on respiration in vitro. If in vitro protoplasms respire to a very similar intensity, irrespective of the size and surface of the animal from which they are derived, Terroine will be right, but if not, then LeBreton's thesis will be justified. Unfortunately, results are not unanimous. In 1924 an in vitro diflference between large and small animals was discovered by Meyerhof & Himwich, and denied by Grafe. In the following year, the former workers were supported by Wels, who found that, without exception, the bigger and older the animal the slower was the in vitro respiration. "Eine bestimmte, vom Nervensystem unabhangige Energiewechselgrosse des Gewebes zu den fundamentalen Arteigenschaften gehort", said Wels. He found, however, that birds had a higher metabolic rate than mammals of equal weight. Fig. 168 constructed from his figures shows the relationships he found. Very similar work was done by LeBreton & Kayser, who got the following figures:
"^ & ""&
c
c. oxygen
taken up per 100 gm.
wet weight per hour, at 37"
Canary
640-7
Mouse
516-7
Rat
343-2
Guinea-pig
2530
Dog
202-7
Roche & Siegler-Soru, thinking that blood would take up oxygen in a more normal manner in the Barcroft apparatus than pieces of tissue, obtained the following figures for the autorespiration of blood :
Oxygen taken up per Calories produced per
Non-nucleated
corpuscles
100 CO
blood per hour
kilo per
Horse
i-i
05
Cow ...
1-26
Pig ...
1-99
08
Sheep
2-07
—
Dog ...
2-15
2-5
Rabbit
2-72
U
Guinea-pig
2-98
756
THE RESPIRATION AND
[PT. Ill
Oxygen taken up per Calories produced per
ucleated
corpuscles
lOOC.C.
blood per hour
kilo per
Turkey
2-86
—
Goose
305
3-5
Duck
3-03
5-0
Hen ...
3-22
5-0
Pigeon
3.78
lO-O
Then Lussana found in the embryo, with its large relative surface, a more intense respiration than in the mother, using the liver and muscle of the guinea-pig, rabbit and goat.
Table 88. Lussana" s in vitro experiments.
Cubic centimetres of gas given off or taken up per lOO gm. wet weight per hour. Foetus at term Maternal organism
Liver
Muscle
Liver
Muscle
Guinea-pig
Rabbit
Dog
Carbon Carbon Carbon Carbon
Oxygen dioxide Oxygen dioxide Oxygen dioxide Oxygen dioxide
1003 1060 205 547 896 918 214 275
2686 2707 283 315 2365 2424 237 273
710 1065 603 835 530 882 833 866
Better figures are those of Kayser, LeBreton & Schaeffer, who compared the oxygen uptake of various tissues according to age, as follows :
Table 89.
Oxygen taken in (cubic centimetres per hour at 37°)
%
Per 100 gm.
Per 100 gm
White Leghorn hens
Embryo water
dry weight
of protein
4 days
94-53
1651
2158
5 days ...
94-52
1090
1546
7 days ...
94-01
940
1380
8 days ...
93-45
747
1118
After hatching
38-day brain .
80-15
1266
2058
2 -year brain .
77-14
823
1543
38-day muscle .
76-31
650
734
2 -year muscle .
67-71
226
249
Pigeons. After hate
ling
2 1 -day brain .
85-13
1 183
1857
I -year brain .
79-83
1035
1739
2 1 -day muscle.
78-65
559
791
I -year muscle .
61-85
302
406
Rats. After birth
5-day brain .
??S
1024
1517
200-day brain .
8qo
1428
5-day muscle .
86-38
812
1075
200-day muscle
74-50
391
443
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
757
These figures demonstrate conclusively that the fall in metabolic rate, which is evident in the case of the intact organism, can also be seen when its tissues are considered in isolation, and can hardly therefore be due to the falling relative surface.
The fact that the oxygen consumption related to loo gm, of protein behaved in the same way as when related to weight was taken by these authors as evidence in favour of their contention that there exist in the cell "albumines de reserve", or paraplasmatic proteins, and therefore that the total nitrogen cannot be regarded as representing the active protoplasmic mass. They regarded the demonstration of LeBreton & Schaeffer, that the chemical nucleoplasmic ratio decreased during development, as a further support for that view. The more paraplasmatic protein present, the less the nucleoprotein in proportion. Finally, they adduced the decreasing watercontent which seems to be so universal an accompaniment of the growth and ageing of protoplasm (cf Ruzicka's "law of protoplasmic hysteresis") as tending in the same direction. As they pointed out, I gm. of dry substance is dissolved or dispersed in 17-2 gm. of water in the 4th day chick embryo, in 3-2 gm. of water at the time of hatching, and in only 2-1 gm. in the adult hen.
Terroine & Roche, on the other hand, investigating the in vitro respiration of tissues, got results which differed from those of Kayser, LeBreton & Schaeffer. They did not concern themselves with the same organism at different ages, but with different organisms of various sizes (homologous tissues of homoiotherms) . Their figures were as follows:
Table 90.
Calories
pro
Cubic millimetres of
oxygen taken up in
vitro per
duced by
intact
gram per
lour
(dry weight)
per hour
Animal
kilo per
Muscle
Liver
Brain
Kidney
Rabbit ...
4
1611
1645
_
Chick ...
5
1603 1636
1484
Guinea-pig
6
1396
2526
I7^5
Pigeon
10
1565
1509
1764
Mouse
20
1601
1764
2025
Finch
37
1532
1511
2476
There was evidently no relation at all between the in vitro respiration of the tissues of the diflferent animals, in spite of their different heatproduction in vivo. It is much to be wished that further work could be done along these lines.
758 THE RESPIRATION AND [pt. hi
4*20. Embryonic Tissue-respiration and Glycolysis
The study of the respiration of embryonic tissues in vitro has taken a great step forward through the work of Warburg and his collaborators, who have related the oxygen consumption of tissues in a very interesting way with the type of metabolism going on in them. Preliminary researches on technique by Warburg and by Minami showed that it was possible to determine on the same material the oxygen consumption per gram per hour and the amount of lactic acid produced both in air and in nitrogen. The lactic acid produced was estimated by using bicarbonate buffer solutions, and calculating from the amount of "extra carbon dioxide" given off^ In the first paper of the series Warburg, Posener & Negelein studied the relations between respiration and glycolysis in a number of tissues. Their basic concepts were analogous to those universally employed with regard to muscle metabolism. In the linked reactions
Glucose — ?► Lactic acid — s-COg and H^O
one of the two may be slower than the other ; the oxidative power of the tissue may be able to remove the lactic acid as fast as it is formed, or conversely, oxidation may be the slower process and lactic acid will tend to accumulate. In intact muscle, as has long been known, the desmolysis and oxidation processes are controlled so that anaerobically lactic acid is formed in great amount, but aerobically it is rapidly oxidised. In chopped muscle, the desmolytic process gets out of control, and nearly as much lactic acid accumulates aerobically as anaerobically; in other words, the oxidation process cannot keep pace with it. By the study of the relative activities of these mechanisms, Warburg and his associates were able to classify tissues to a considerable extent. In this work they used three symbols, defined as follows :
^ c.mm. of Oo used up , • ^- \ r, n
Q^o, = 7^^^ r-^- — respiration) R.R.
mgm. of tissue x hours
0°^ = c-mm. extra GO, given out in O, (^..^bic glycolysis) O.G.R. ^co, mgm. of tissue x hours
„ N^ ^ c.mm. total ^ CO, given out in N, (anaerobic glycolysis) ^coa mgm. of tissue x hours N.G.R.
^ Assuming the R.Q.. of the tissue to be unity.
^ Anaerobically there can be no "extra" COj because respiration in the sense of oxygen-consumption is not proceeding; all of it must be due to lactic acid formation.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 759
For convenience the three symbols adopted here will be R.R. (respiratory rate), O.G.R. (aerobic glycolysis rate), and N.G.R. (anaerobic glycolysis rate), i c.mm. of extra carbon dioxide (from the bicarbonate) corresponds to 0-004 mgm. of lactic acid, Warburg, Posener & Negelein made many experiments to delimit accurately the effect of pH, concentration of bicarbonate buffer and glucose, temperature, presence of serum, etc., on the three entities: thus with increasing j&H (6-7 to 7-8) the N.G.R. rose from about 7 to about 16. But what more directly interests us is their comparative results for different tissues, in the estimation of which they were careful to observe standard conditions (37-5°, 0-2 per cent, glucose, 2-5 x 10-^ % bicarbonate, pH 7-66). They noted here yet a further entity, namely,
^, ,^ , r- ■ lactic acid disappearing N.G.R. - O.G.R.
the Meyerhof-quotient -. r^-^- ^ or — —
respiration R.R.
(M.Q^.), which gives a measure of the extent to which the lactic acid formed is built up again into the glucose or the hexose-phosphate.
The results of their experiments are given in Table 9 1 . A glance at it shows several very important results. In the first place, the Meyerhof-quotient is normal for all tissues studied, a fact which Warburg regarded as evidence that the desmolysis mechanism, though working unduly intensely in certain tissues (especially the neoplasms), was normal in its nature. In every case, moreover, the rate of desmolysis of carbohydrate, or rather the rate of accumulation of the lactic acid, is higher anaerobically than aerobically, but tissues vary a great deal in the extent to which this is so. In some cases, the percentage inhibition of lactic acid accumulation which occurs when the tissue passes from anaerobic to aerobic conditions is great, perhaps as much as 95 per cent. But in other cases, and these include the neoplasmatic tissues, the inhibition due to the letting in of oxygen is only small. Thus the rat carcinoma has a glycolytic power 1 24 times that of adult blood, 200 times that of resting frog muscle, and 8 times that of acting frog muscle. As for the respiratory intensity, it varies quite independently of the other entities, and is sometimes large, sometimes small. As the extreme instance of the glycolytic tissue, Warburg cited yeast, which desmolyses large amounts of sugar aerobically as well as anaerobically. As the extreme instance of the respiratory tissue, there is Pasteur's mould, Mucor mucedo, and the table provides an example, rabbit pancreas or submaxillary gland, for instance, allowing no lactic acid at all to accumulate aerobically. It was obviously
76o RESPIRATION AND HEAT-PRODUCTION [pt. iii
very important that Warburg found the tissue of neoplasms to behave unUke muscle and like the yeast-cell. But it is more interesting for the present purpose to note that he found the chick embryo (from the 3rd to the 5th day of development) to be different alike from adult and from neoplasmatic tissue. In its efficiency at removing lactic acid when allowed air, it resembled muscle, but its general metabolic level was of course higher and appeared in the big R.R. The relation between aerobic glycolysis (O.G.R.) and R.R. is also interesting; thus from the last column it appears that the chick embryo produces only o-i mol. of lactic acid for every mol. of oxygen taken in — a very different state of affairs from the tumourcells, which will produce as much as 3-9 mol. of lactic acid for every mol. of oxygen, though it is not unlike the adult tissues, which occupy an intermediate position. Warburg found that by adding a trace of hydrocyanic acid to the medium containing the embryonic tissues he could, as it were, put a spoke into the wheels of the oxidation mechanism, and bring about a state of affairs resembling that of tumours. Thus he was able to send up the O.G.R. of the 4th-day chick embryo from i-i to 12-0, to bring down the percentage inhibition from 96 to 45, and to make the embryo produce 3-4 mol. of lactic acid for every mol. of oxygen taken in. This was a good imitation of a malignant carcinoma. Moreover, tumour-cells + hydrocyanic acid gave the same O.G.R, as N.G.R., showing that oxidations had been entirely depressed. The benign tumour-cells, with their O.G.R./R.R. of about 0-9 could, he found, be equally well imitated by incubating embryonic tissue anaerobically for some time before beginning the experiment. But though he was able thus to induce in embryonic material the characteristics of neoplasmatic tissues, he was not able to reverse the process, or to ascertain how it was that these characteristics were retained for a great length of time by some cells. Such observations as these acquire no Uttle significance from the fact that sarcomata can be produced in adult animals by injecting embryo pulp with arsenious acid (Carrel; White; Askanasy; Mcjunkin & Cikrit; denied by Begg & Cramer) with tar (Carrel) and with indol (Carrel).
The metabolism of the neoplasms was called by Warburg that of disorganised growth, and the metabolism of the embryo that of organised growth. These distinctions are of interest in view of the work of Byerly (see p. 607), and have a relation to much recent
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764 THE RESPIRATION AND [pt. iii
investigation of differentiation-processes. Referring again to Table 9 1 , it will be found that embryonic tissue has a very considerable lactic acid production in anaerobic conditions, as much as that of the neoplasms, but that it differs from them on the one hand by the efficiency of its aerobic oxidation mechanism (40 times as adequate as that of rat carcinoma), and from adult tissues, on the other hand, because of their feeble anaerobic glycolysis rate. Per hour per milligram wet weight the 4th day embryo can produce anaerobically 0-09 mgm. of lactic acid, i.e. 9 per cent, of its weight; this may be set against the performance of frog's muscle (intact and anaerobic) which produced only o-o6 per cent, of its weight when resting, and 1-5 per cent, when stimulated. Warburg concluded that the high N.G.R. was a general property of growing tissues, but that the O.G.R. was only high if the growth was unorganised as in neoplasms, and regarded this as the most important outcome of his work. "Where growth is, glycolysis is ", he said, " and where abnormal growth is there aerobic glycolysis is." As regards the adult tissues he tried, there were one or two difficulties. Those that might be considered stationary, such as liver and kidney, had very low N.G.R.'s and unmeasurably small O.G.R.'s, while those which were not quite rightly termed stationary, such as testis, had slightly higher N.G.R.'s and small O.G.R.'s. But the grey substance of the brain and the retina were found to have quite peculiar characteristics, an enormously high R.R., nearly three times that of the chick embryo, a very high N.G.R. and a high O.G.R. No satisfactory explanation was or is available for this curious state of affairs, but it has its importance for embryological studies, since in the earlier periods of development the brain vesicles and the eyes make up so significant a proportion of the growing body. The possibility that these results might explain some of those obtained by Child and his school must not be forgotten. Subsequently Negelein found quite different results with amphibian retina, and suggested that such a delicate tissue had been giving cytolysis results in the earlier work. But more recent researches by Krebs have confirmed it.
Finally Warburg, Posener & Negelein gave some interesting data on the subject of ammonia production, glycolysis and tissue respiration. They found that, when the tissues were placed in Ringer solution to which glucose had not been added, notable amounts of ammonia were formed, indicating a combustion of protein substances. They
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
765
compared the activities of various tissues in this direction, obtaining the following results :
Rat thyroid
Rabbit submaxillary
Rat liver
Rabbit pancreas ...
Rat thymus
Rat testis
Chick embryo (5th day)
Rat carcinoma
Rat grey matter ...
Rat retina ...
Cubic millimetres of ammonia produced (in glucosefree Ringer) /milligrams of
tissue X hours N.G.R.
o 2
0-03 3
0-07 3
on 3
0-31 8
003 8
0-56 23
0-9 31
1-4 . 19
It is evident that all the tissues concerned prefer to combust carbohydrate if they can get it, but if not they will combust protein. It would be very interesting to know how this property varies during development in the chick. A certain parallelism is to be observed between the anaerobic glycolysis rate (not the R.R.) and the ammonia-production rate in aerobic glucose-free conditions. The addition of glucose abolished the ammonia production both aerobically and anaerobically.
The next important paper was that of Negelein, who devoted himself particularly to the examination of the changes taking place in respiratory characteristics during embryonic growth. Chick embryos, as Warburg, Posener & Negelein had already found, produced anaerobically 9 per cent, of their weight in lactic acid per hour; rat embryos, Negelein now found, produced only between 3 and 7 per cent. But all depended on the time of development, for in the earlier stages rat embryos had a figure of about 14 per cent. The amniotic membranes and the chorion were found to have an even larger figure, as much as 19-6 per cent., i.e. fully as high an N.G.R. as neoplasms. The curve which resulted from Negelein's observations appears in Fig. 169. The measurements were nearly all
^30
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—
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i
',
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s
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1
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Trockengewichteines Embryos (mgr) Fig. 169.
766
THE RESPIRATION AND
[PT. Ill
done in inactivated horse serum, though in later stages amniotic liquid was used. For comparison some of the figures are included in Table 91 . The curve obviously declines with increasing age, the N.G.R. falling from about 35 to about 5, so that, as can be seen from Table 9 1 , the N.G.R. declines during the embryonic period of the rat from a value equivalent to that of malignant neoplasms and to the very young chick embryo to a value very close to that of the various adult rat tissues. According to one or two of the earhest measurements of Negelein, there is a possibility that the high value of 35 at 0-5 mgm. dry weight may be a peak, to which the curve has risen from earlier lower values. If this turns out to be the case, there will be an interesting parallel with the hen's egg, which, according to Tomita's observations, has a marked peak in lactic acid content at the 5th day of incubation (see Fig. 292). Negelein argued that the unfertilised egg-cell has probably only a very small N.G.R., so that a peak would be expected. That it comes so early in development is a fact of importance from the point of view of the energy source used by the embryo. Negelein found that the N.G.R. of the chorionic membranes of the rat embryo decHned in much the same way as that of the embryo itself This is shown in Fig. 170, where the age of the membranes, expressed in embryo dry weights, is plotted against the N.G.R., and a few figures are given in Table 91. It is noteworthy that the N.G.R. of the membranes is always much higher than that of the embryo, although it falls in unison with it, if a Httle slower. It maintains its N.G.R. at a level well above that of most neoplasms, so that its hydrolytic mechanisms must be exceedingly powerful. Negelein only did one experiment with the amniotic membrane separately; it gave an N.G.R. of 35-8 at an embryo dry weight of 60 mgm., i.e. slightly above the corresponding N.G.R. of the chorion.
Negelein's rat embryos gave rather variable results with respect to the O.G.R., for in Ringer solution a measurable amount of lactic acid was formed aerobically, but in serum this only occurred in the
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SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
767
early stages. Negelein's conclusion was that in the best conditions there was practically no O.G.R., a finding which agreed very well with the previous results of Warburg, Posener & Negelein on the chick embryo.
In 1927 Warburg collected together the data which had accumulated for various tissues, with the object of proposing another entity, the fermentation excess, or U., which he defined as equal to N.G.R. — 2 R.R. Previously the comparison of tissues on the basis of their O.G.R. had been difficult because of the high suscepdbility of the reaction on which it depended; thus rat embryos in Ringer had a considerable O.G.R. but in serum only a very small one, while in amniotic fluid they had none at all. He therefore suggested that it would be best not to measure the O.G.R. directly, but to calculate it from theoretical grounds. U. would be o when the N.G.R. equalled double the R.R., and negative or positive if it was lower or higher than double the R.R. In the latter case, the oxidation mechanisms would be inferior in efficiency to the glycolytic mechanisms and vice versa. Values for U. are given in Table 9 1 .
Other workers continued the investigations in the matter of embryonic tissues. Krebs discovered the interesting fact that the adult bird retina had a metabolism almost entirely composed of glycolysis, with an almost unmeasurable respiration. His figures are shown in Table 91. At the same time he found that the embryonic retina had not so extreme a type of metabolism, having an O.G.R. of 33, unlike the value of 133 for the adult. The construction of a curve relating avian retina N.G.R. to age was undertaken by Tamiya, whose graph is shown in Fig. 171. Beginning on the 8th day of development at about 35, it rose steadily during incubation to reach 45 at the 15th day and 85 at hatching, after which it continued to rise during postnatal life, until the adult value of 130 was reached. Some observations seemed to show that in old age it declined again, and was 90 at 4 years old. This result implied that just before
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768
THE RESPIRATION AND
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hatching the embryonic retina produced anaerobically 20 per cent, of its weight per hour lactic acid, but that i year after hatching it produced under similar conditions in the same time 65 per cent, of its own weight. This tissue has certainly a far greater glycolytic intensity than any other known, and Cramer suggests that this peculiar property may account for the fact that human epitheliomata and neuroepitheliomata of the retina have a very short induction-period unlike all other neoplasms. On the other hand the em
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bryonic lens of the rat was shown by Fujita to have a constantly descending N.G.R. (see Fig. 172).
Tamiya later made further experiments on the developing liver of the chick. His graph is shown in Fig. 173. It resembles the declining glycolytic rate of the rat embryo, and differs entirely from the behaviour of the retina. U. calculated for each stage was negative.
20
0,1 0,1 0^ 0,f 0,5 0,6 0.70,8 0,9 1,0 1,1 1,2 1,3 f^ 1,5 1,6 7,7 1,8 1,3 2,0 2,1 2,2 2,3
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Fig. 173.
i.e. O.G.R. was very low, and the liver-cells at all times during their growth were able to deal with as much lactic acid as their desmolytic mechanism supplied them with. Their metabolism was throughout that of well-organised growth, like that of the rat embryo. Similar work was done by Warburg & Kubowitz on embryonic fibroblasts, epithelial cells and heart cells. The figures are given in Table 91. For the heart, their results agreed well with those of Tamiya for the embryonic liver. As age increased, and the weight
A
1
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
769
of the heart (dry) rose from o-i to o-8 mgm., so the N.G.R. fell from about 50-25 to 14. It is interesting to note that, in their figures for tissue cultures of fibroblasts, the R.R. and the N.G.R. were both systematically higher the fewer the number of sub-cultures that had been gone through since the tissue was first explanted. Perhaps this might be regarded as evidence that with increasing age the metabolic rate declines in tissue culture, just as the growth-rate does, as was shown by Cohn & Murray (see p. 461 and Figs. 62 and 174).
Work on embryo tissues was further extended by Kumanomido, who studied the respiratory metabolism of chick embryos in chick serum and in Ringer solution. The N.G.R. was in all cases some 50 per cent, higher in the latter than in the former. Kumanomido felt able to conclude that this difference was not due to a cytolysis or other injury effect
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No.of times the fibroblasbs were subcultured
Fig. 174.
in Ringer, but to an inhibiting effect of serum, owing to which the maximum intensity of lactic acid production was not reached there. Dialysis of the serum so that most of the salts and crystalloidal substances disappeared did not affect its depressant property, nor did heating for 30 minutes at 55°. Kumanomido did not state, however, whether these conclusions applied to the O.G.R. as well, but it would be important to know this, in view of Negelein's results on rat embryos, where there was a measurable O.G.R. in Ringer but not in serum. Fig. 175, taken from Kumanomido, shows the decline in N.G.R. with age during incubation in the chick embryo. This graph is directly comparable with that of Negelein for the pre-natal life of the rat, shown in Fig. 1 69 and with that of Rosenthal & Lasnitzki for the developing liver and kidney of the rat shown in Fig. 1 76.
The extent of the fall in anaerobic glycolysis rate does not seem to be so great in the chick as in the rat, for in the former the drop is from 27 to 10, and in the latter from 35 to 5. But Kumanomido's
770
THE RESPIRATION AND
[PT. Ill
chick embryos were all taken during a very short period, namely the 2nd to the 5th day of incubation, and it is possible that a more complete study would reveal a greater fall. In Table 9 1 it will be noted that, following the usual course, U. is always negative, and, though the N.G.R. is fairly high, the O.G.R. is low. The R.R. shows some indications of a fall with age, agreeing with other figures for metabolic rate.
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It appears that in general the N.G.R. declines with age^. This has been found to be true in several investigations :
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Rat kidney
Rat embryo (whole)
Rat chorion
Human placenta
Chick liver
Chick heart {in vitro)
Chick embryo (whole)
Rat lens and placenta
Rat placenta
Rosenthal & Lasnitzki
Rosenthal & Lasnitzki
Negelein
Negelein
Loeser
Tamiya
Warburg & Kubowitz
Kumanomido
Fujita
Adler
The converse, i.e. that the N.G.R. rises with age, has only been observed in one case :
Chick retina ... ... Tamiya
The aerobic glycolysis rate, on the contrary, may either rise or fall with age;
Rosenthal & Lasnitzki
Loeser
Kumanomido
Rise: Rat liver
Fall: Human placenta
Chick embryo (whole)
^ Fertilisation, according to Ashbel, augments the N.G.R. of echinoderm
eggs.
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO
771
Rosenthal S^Lasnibski (Rat) ver kidney • ♦ NGR
O OGR
It would seem, therefore, that Hawkins is not very wide of the mark when he suggests that N.G.R. is a function of growth-rate. He believes that tissues can be classified better in this way than by using R.R. and O.G.R. relationships as the German school does.
Kumanomido also made a few experiments with the chorion of hen and rat embryos and with rat embryos themselves, finding in all cases that the N.G.R. was higher in Ringer solution than in serum. He examined a few normal human chorionic membranes and placentas, and obtained a very low N.G.R. (of about 5). There was here a certain contradiction between his results and those of Murphy & Hawkins, who worked with rat placentas, and got the results shown in Table 9 1 . However, neither of these papers contain details of the age of the placentas used, though from Kumanomido's description it would seem that he used fullterm material. Murphy & Hawkins' paper was mostly concerned with the in vitro respiration and glycolysis of various types of neoplasm, but they confirmed the results of Warburg, Posener & Negelein on the chick embryo, and stated that the same type of respiratory metaboHsm was shown by rat embryo skin, rat embryo membranes, and the wall of the pregnant rat uterus, although they gave no figures in support of this.
The most illuminating suggestion as regards the placenta in this connection is that of Bell, Cunningham, Jowett, Millet & Brooks, who found, as is shown in Table 9 1 , that the early human placenta gave a positive U., i.e. possessed on the whole a more active glycolytic than oxidative mechanism. Then, removing the chorionic epithelium, they made the experiment again, and found that the result was now a negative U. like that given by practically all non-neoplasmic tissues. They therefore did not hesitate to regard the penetration of the maternal tissue by the invading foetal trophoblast as truly "malignant", a standpoint of much interest from several points of view
2 34 5 6 78 9101112 1 cms. length of body Adult
Fig. 176.
772 THE RESPIRATION AND [pt. hi
(see also Superbi) . Thus Hammond who implanted foetal tissues into the uterine wall, found that they possessed no power of establishing themselves there, unlike the foetal trophoblast. The uterine grafts were absorbed.
Summarising, we may say that four main types of respiratory metabolism have been revealed by the studies initiated by Warburg :
(a) Normal resting tissue — anaerobic glycolysis slight, aerobic glycolysis absent, respiratory rate generally rather high, U. negative, Warburg quotient ^ 0-3.
{b) Normal growing tissue [embryonic) — anaerobic glycolysis high, aerobic glycolysis very slight or absent, respiratory rate high, U. negative, Warburg quotient ^ 0-3,
[c) Abnormal growing tissue {malign neoplasms) — aerobic and anaerobic glycolysis both high, respiratory rate usually low, U. positive, Warburg quotient > 2-0.
[d) Abnormal growing tissue {benign neoplasms) — all factors generally rather low, U. positive, Warburg quotient 0-5-1 -3.
Completer details are to be found in the reviews of Warburg & Minami ; Warburg, Negelein & Posener ; Cramer ; Cannan ; and particularly Warburg.
4-21. The Genesis of Heat regulation
The only other subject which calls for consideration in this chapter is that of the ontogeny of heat regulation. It has been known for a long time that, just as animals which are normally homoiosmotic are yet poikilosmotic while in their embryonic state, so the capacity of heat regulation possessed by homoiothermic organisms arises at a definite moment in the individual life-cycle.
Edwards, who worked about the year 1820, was the first to notice this. He found that the temperature of newly born puppies, kittens, and rabbits fell when the animal was removed from its warm surroundings, and continued to fall until it almost reached the temperature of the air. Guinea-pigs, on the other hand, were able to maintain their temperature very well immediately after birth. Edwards divided homoiothermic animals into two classes, {a) those which are at birth bhnd, helpless, naked, and poikilothermic, and {b) those with open eyes, skin covered with hair or feathers, and homoiothermic. This classification corresponds exactly with nidifugous and nidicolous birds (see pp. 272 and 317). In the case of
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 773
the dog, the cat, and the rabbit, the adult condition is reached 15 days after birth. Edwards found that these phenomena were not due to the surface/volume ratio of the small animal, and showed that skin covering had little to do with it, for an adult sparrow was able to maintain and regulate its temperature perfectly in the absence of all feathers. Many years later Raudnitz, from a study of the temperature of newly born human infants, concluded that the chief cause of variable temperature was the imperfect development of homoiothermicity.
Babak, continuing the analysis of the problem, divided heat-regulation into two types, ' ' chemical ' ' and ' ' physical ' ' . The human newborn infant, for example, possesses the former function, for it can counter a fall of environmental temperature by increasing its combustions ; but it cannot resist a rise and immediately goes into hyperthermia, having an imperfect control of its capillaries and its transpiration. Plant subsequently extended this point of view to other mammals such as the cat and dog.
The bird embryo was first investigated in this connection by Pembrey, Gordon & Warren. They demonstrated that, during the incubation period in the chick, changes of external temperature invariably determine, after a longer or shorter period, changes in the same direction in the respiratory exchange. The period required for response did not seem to depend on whether the shell was removed or not. On the other hand, when the recently hatched chick was examined, it reacted to temperature changes exactly as a warmblooded animal would, a fall of 20° in external temperature raising the expired carbon dioxide to twice its previous amount in 15 minutes. The chick embryo, then, was cold-blooded up to the 19th day of incubation. The physiological transition was observed by Pembrey and his associates to take place on the 21st day of incubation during the time immediately prior to hatching. Sometimes there was an intermediate condition, transient but neutral, in which a fall of temperature did not result in a fall in carbon dioxide output, nor, on the other hand, in a rise. This intermediate condition may give way to the cold-blooded or the warm-blooded condition, according as to whether the chick is feeble or strong and healthy.
Pembrey later reported that the transition to full powers of heat regulation did not take place in the pigeon (a nidicolous bird) till well after hatching, i.e. about the 6th day of post-natal life. Giaja
774 THE RESPIRATION AND [pt. m
was the next investigator who occupied himself with this problem.
Giaja defined the "summit metabolism" as the maximum energy
expenditure which the homoiothermic animal can rise to in its
struggle against cold, and spoke of a "metabolic quotient" as
follows: ^ . ^ 1 r
Summit metabolism , _
— n z r-rr-p = metabolic quotient.
Basal metabolism ^
For the mouse and the rat the metabolic quotient is approximately 3*5, for birds 4-0. Before hatching, the chick embryo, according to Giaja's measurements, which were quite comparable with those of Pembrey, could be said to have a negative metabolic quotient, but by 6 hours afterwards the value of this constant was i-g, and, 5 days after, it rose to 2-0, and at 48 days to 2-6. Giaja observed the intermediate state spoken of by Pembrey, i.e. the condition before hatching, in which the chick maintains its heat-production when the temperature is lowered but cannot raise it to compensate for a fall. Exactly the same results were obtained on the rabbit immediately after birth; its metabolic quotient, 1-3 at 12 hours, rose to 2-4 a fortnight later.
A certain further insight into the ontogeny of heat regulation is obtained by examining Fig. 83 b, which has already been described. It shows Brody & Henderson's work on the effect of temperature on the growth-rate of the chick embryo at different stages. The reason why the curve X^ is not a curve but a straight line is, in their opinion, that between the 13th and i8th days of incubation the heat-regulating mechanism of the embryo has probably developed sufficiently to enable it to keep its body-temperature constant within the limits of 37-2° and 40-6°. Thus at this stage the chick embryo is not strictly cold-blooded. These experiments would authorise us to suppose that the development of heat regulation is not quite sudden, as it seemed at first to be from Pembrey's experiments, but the zone of temperatures within which it can accurately adjust itself widens continually as development progresses. The most recent investigations of this are those of Kendeigh & Baldwin. They state that "irequently in the literature of avian physiology and life-history, statements are to be found that the body-temperatures of nestling birds are extremely variable and similar to those of cold-blooded animals". Their own work on the house wren, Troglodytes aedon, showed clearly that full temperature regulation was attained by the
SECT. 4] HEAT-PRODUCTION OF THE EMBRYO 775
time that the squabs were ready to leave the nest. They distinguished four factors in the installation of this function, (i) the change in the surface/volume ratio, (2) the development of feathers, (3) the development of an internal dissipating surface (the air-sacs) probably under nervous respiratory control, and (4) heat-production in metaboHsm. Of these they regarded the third as perhaps the most important.
In a later paper Pembrey found that mice and rats at birth have not attained the capacity for heat regulation. When they are naked, blind, and unable to run about, they cannot maintain a constant temperature, and cannot regulate the production of heat at low temperatures. When they are about 8 days old, they have a protecting coat of fur, and though still bhnd are much more active — at this stage they give some evidence of having acquired the power. At 10 days old they are as homoio thermic as the adults, Gulick's later statement that mice and rats are fully homoiothermic at birth cannot be accepted on the basis of his insufficient evidence. Exactly similar results were obtained on pigeon squabs, as has already been mentioned, so that the pigeon and the hen among oviparous animals correspond respectively to the rabbit and the guinea-pig among viviparous ones. More recently, Ginglinger & Kayser have confirmed the work of Pembrey and have linked up, as we have already seen, the genesis of heat-regulation with the peak in metabolic rate. Their results may be summarised as follows :
Form of heat-regulation present at birth
Chemical Physical
Guinea-pig . .
Chick
Rabbit
Man
Cat
Mouse
Pigeon
It may not be irrelevant here to mention, in connection with body-temperature, that the so-called "broody fever", i.e. a temperature higher than normal for sitting hens, has been shown by Simpson to be without factual basis.
776 RESPIRATION AND HEAT-PRODUCTION [pt. iii
4-22. Light-production in Embryonic Life
Heat is, of course, not the only form of energy which animals can radiate. Luminous organisms occasionally have luminous eggs as the following table shows :
COELENTERATA
Ctenophores
Beroe. Segmentation-stages, not the eggs
Allman; Agassiz, A.; Peters, A. W.
Annelids
Polychaeta
Chaetopterus. Larvae
Enders
ECHINODERMS
Ophiuroidea
Plutei
Mangold, E.
Arthropods Urochorda
Crustacea Insects
Schizopod larvae Copepod nauplii Coleoptera. Lampyris
Pyrophorus Tunicates. Pyrosoma
Trojan
Giesbrecht
Harvey
McKinnon
The beetle's eggs in question shine before fertilisation, while still in the ovary. It would be interesting to know what component is missing from the early developmental stages of Beroe. In the case of Pyrosoma, according to McKinnon, the luminescence is due to the presence of symbiotic bacteria which are transmitted to each egg as it is laid by a special mechanism in the parent and are afterwards distributed among the blastomeres during the cleavage stages. Similar events take place in certain cephalopods which produce luminous eggs.
McKinnon also points out that other symbiotic organisms may be transferred to eggs. Thus the reinfection of the termites such as Hylecoetus dermestoides with fungal spores is brought about by the smearing of the spores on to the egg as it passes down the oviduct and the subsequent consumption by the larva of its own egg-case after hatching. The wood-wasp Sirex and the olive-fly Dacus are other instances of this. Moreover, the transmission of the symbiotic cellulose-fermenting yeasts from generation to generation occurs in a similar way in various beetles (the death-watch beetle, Xestobium rufovillosum (Staniland); Sitodrepa panicea (Breitsprecher)), also in a large number of homoptera (Richter) and in the rice-weevil, Calandra oryzae (Mansour). The whole subject has been well reviewed by Buchner.
Cite this page: Hill, M.A. (2024, April 27) Embryology Book - Chemical embryology 2-4 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-4_(1900)
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