THE RELATIONS BETWEEN YOLK AND WHITE
IN THE HEN'S EGG
I. INTRODUCTION
BY JOSEPH NEEDHAM AND MICHAEL SMITH
(From the Biochemical Laboratory and the Low Temperature Research Station, Cambridge.)
(Received 8th May, 1931.)
NOTHING can be more characteristic of the physical basis of life than the separation
by membranes of phases differing greatly in their constitution and concentration both of organic and inorganic compounds. The prominent part played by membrane phenomena in general physiology is therefore not surprising.
Relations between Yolk and White in the Hen's Egg 287
A further illustration of the remarkable properties of this membrane in its natural surroundings has been given since Straub's work. According to Chick, Copping and Roscoe (6), vitamin B2 is contained in the egg-white, but not B^ while
the yolk contains both B2 and Bx. If, however, undiluted yolk was placed in a
collodion bag, with water outside, the Bx factor escaped into the water. And an
artificial system constructed of a cellophane membrane and watery yeast extracts showed that Bx and B2 passed through the barrier and attained a state of uniform
distribution.
Straub entered a new field when he suggested that the osmotic difference between yolk and white was a " Lebenswirkung " in the sense that the membrane might actually do work thermodynamically in opposing the dilution of the yolk. If this were so, the infertile egg might be expected to show after long storage a disappearance of the membrane properties which characterised it when quite fresh. He mentioned in this connection the experiments of Wolff (29), who had found that in the fresh hen's egg there is a trace of zinc in the yolk and a trace of copper in the white, but that after storage for some time, these metals are equally distributed throughout the egg. Straub showed that eggs stored for a long time do tend to acquire uniformity of osmotic pressure on both sides of the membrane at about — 0-50° C. He also stated that the injection of small amounts (2 or 3 mg. per egg) of morphine, cocaine, or potassium cyanide, would interfere with the function of the membrane and lead to an unusually rapid equilibration. But Straub's two most important experiments were these: (1) he put yolk into a parchment capsule with egg-white outside and found that the system quickly attained osmotic equilibrium (24 hours); (2) he placed the yolk of a fresh egg in diluted egg-white, and after leaving it there for some hours, replaced it in ordinary white, whereupon its osmotic pressure, which had been lowered, rose above, not merely to the same level as, that of the ordinary white. Thus in all situations the yolk maintained its natural hypertony to the white. Straub concluded that the "living" vitelline membrane tended to encourage the exit of water from the yolk or to impede its entry, on the one hand, or to encourage the entry of salts and impede their exit, on the other hand. And this behaviour was exhibited not only in the intact egg suffering slow change over a long period of time, but also in the strict reversibility of a yolk placed in dilute egg-white for 24 hours. It may be said here, however, that the following papers of this series will give confirmation only to the first of these two experiments.
weaker. Similar relations are found in worms such as Gunda ulvae, according to Pantin(zi). And Nassonov(i9) arrives at the category of osmotic work by his demonstration that anaerobiosis leads to profound modifications in the vital staining of cells (degranulation, nuclear stain, etc.), and by his conception of " Verteilungs-energie," work done to maintain the distribution of materials characteristic of the normal cell. But all these cases involve the presence of living cells, and what is so interesting about the vitelline membrane of the hen's egg is that it is not cellular, except in so far as the whole yolk is regarded as a living cell. It provides a very large-scale system for the examination of the mechanism of such osmotic work.
The actual magnitude of the concentration work done at the surface of the hen's-egg yolk was calculated by Straub to be o-oi gm. cal. per egg per day. Now there exist in the literature one or two investigations of the gas exchange and heat production of infertile eggs. Stepanekta) found that one unfertilised egg gave off 10 mg. of carbon dioxide per day at 390 C , and Langworthy and Barott(i4), using large numbers of eggs, observed a heat production of o-oi gm. cal. per kg. per hour at 12° C , 0-02 at 15° C. and 0-06 at 19° C. Then Pucher(zz) studied the changes taking place in an incubated infertile egg during 20 days from laying. He found that the total glucose of the white fell by 90 mg. per cent, and the total glucose of the yolk rose by 40 mg. per cent. For purposes of rough calculation the white may be taken as 33 gm. and the yolk as 16, in which case the former loses 30 mg. in 20 days and the latter gains 6 mg., giving a total loss from the whole egg of 24 mg. or i-2 mg. per egg per day. This was confirmed by Ido(n), who incubated fresh hen's eggs at 37° C. anaerobically for 5 weeks and observed a decrease in the free glucose of the whole egg from 352 to 279 mg. per cent., i.e. about 29 mg. per egg or 0-83 mg. per egg per day. The greater part of this loss occurred in the first two weeks, however, so this figure must be on the low side. Langworthy and Barott's value for heat production amounts to 0-072 gm. cal. per egg per day. All these quantities, it may be noted, are considerably larger than what would be required for the concentration work calculated by Straub, i.e. a quota of energy derivable from the combustion of 0-0025 mS- glucose daily. Straub gave an interesting theoretical excursus suggesting physical mechanisms by means of which the energy derived from this process could be used at the surface of the vitelline membrane. He regarded it as a galvanic combustion element for glucose with oxygen to carbon dioxide and water, so that the maintenance of the steady state in the hen's egg would be a complicated case of concentration polarisation.
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without could readily liberate. In the third paper of this series, new data will be presented in an attempt to answer the question whether the infertile hen's egg shows any measurable respiration (oxygen uptake).
If Straub's view were correct, there should be a continual, though small, oxidation of hexose to carbon dioxide and water in the infertile egg, so that under anaerobic conditions the mechanism would cease to function and, energy being no longer available for the membrane, dilution of the yolk would take place. It was left for Hill (9) to make the simple experiment of placing fresh eggs in hydrogen and observing the effect on the osmotic pressure difference between yolk and white. Osmotic pressure was not measured directly in Hill's experiments, but was calcu-lated from the vapour pressure, itself derived from the evaporation rate of the solution in question (yolk or white) determined by a very delicate thermopile in contact with filter paper saturated with the solution (see Margaria (17)).
By this means it was found that the vapour pressure difference between yolk and white became smaller at the same rate whether the eggs had been in air or in pure hydrogen. If, as Hill said, a continual liberation of energy is the means by which the egg evades the attainment of osmotic equilibrium between yolk and white, that energy can be obtained from some source not involving the use of free molecular oxygen. This conclusion recalled the almost forgotten work of Stepanek (25), who had shown, at any rate to his own satisfaction, that hen's eggs, incubated anaerobically with glucose solutions, would produce considerable amounts of lactic acid and alcohol. As far as the production of lactic acid was concerned, Stepanek had been subsequently confirmed by Tomitato) and IdodO, while Aokiw reported an extremely small accumulation of alcohol in intact eggs. The possibility was thus open that the essential reaction for the osmotic work might be a glycolysis.
osmotic properties of the intact yolk suspended in artificial solutions are very complicated.
From histology it seems that little assistance can be had (e.g. LecaillonOs)), for when the membrane does not appear quite structureless, all that can be seen is a network of keratin fibres.
It is, of course, open to question whether the comparatively rigid keratin envelope around the yolk is really the active system in the maintenance of the egg's steady state. It is usually assumed by morphologists that a protoplasmic layer exists beneath the vitelline membrane in the infertile egg, but, as far as we know, there is no evidence for this except the purely analogical comparison of the avian yolk with such a system as the echinoderm egg cell. The case of the yolk, it is urged, may be similar to that of adipose tissue cells, where no investing protoplasm can be seen until the fat has been dissolved away. It is also stated that the yolk as originally laid down is intracellular, being associated with the Golgi bodies. Perhaps the burden of proof that a protoplasmic membrane exists underneath the vitelline membrane rests with those who affirm it, and it is for consideration whether a simpler and equally adequate explanation of the facts may not be possible without it. Again, some authors speak as if the yolk was permeated throughout by protoplasm, but if this were so, it should give some measurable respiration in vitro, and, as will be shown in the fourth paper of this series, it does not. It is true that the cells of the growing blastoderm have their own cell membranes, which are obviously distinct from the vitelline membrane (edges of blastoderm in section show this well), but in the absence of any evidence to the contrary it seems reasonable to assume that in the infertile avian egg the cell is a relatively small protoplasmic mass underneath and in the centre of the germinal area, containing the nucleus or germinal vesicle of Balfour ((3), 2, 121). The granular material of the germinal disc may of course respire, but an experimental test of this would be difficult. On this view, the yolk material, generated originally in connection with the Golgi bodies in the cell, has been extruded into the space within the vitelline membrane.
A word may be said concerning the bound water in the yolk and white, which, if defined as the water in which added solutes will not dissolve, might be of importance in the osmotic equilibria with which the vitelline membrane has to deal. Hill do) has assessed experimentally the bound water in the infertile hen's egg as 3 per cent, of the water of the white and 15 per cent, of the water of the yolk. This means that in an average egg the white would contain 22-9 gm. of free and 0-71 gm. of bound water, while the yolk would contain 60 gm. of free and 1 -05 gm. of bound water. The amounts of bound water on each side are then roughly the same.
The case of the trout egg is not entirely parallel with that of the hen, but it is interesting that, according to Gray, the osmotic pressure of the contents can be kept up for a long period under anaerobic conditions (7). If any work is done it must be anaerobic, but unfortunately we know nothing about the glycolytic mechanisms of the trout egg.
Relations between Yolk and White in the Hen's Egg 291
A difference of osmotic pressure amounting to between 1 and 2 atmospheres exists between yolk and white in the freshly laid hen's egg.
This difference is probably of great embryological importance because of its relation to the water current yolkwards which occurs during incubation of the fertile egg.
At room temperature this difference persists for at least two months, diminishing slowly in value, and to maintain it it is possible that work must be done at the vitelline membrane, preventing the dilution of the yolk.
If so, the mechanism providing the energy for this work continues to function anaerobically. Furthermore, unfertilised eggs produce carbon dioxide in con-siderable amounts, and lose a small quantity of glucose (aerobically, Pucher(22), anaerobically, Ido(n)). They also evolve an extremely small amount of heat, and this varies with the temperature.
Both lactic acid and alcohol are present in the fresh infertile egg (Tomita(27), Aoki(2)), and the latter increases in amount as the egg ages at room temperature (Aoki (2)). Incubated with glucose solutions, the egg produces lactic acid (yolk alone, Tomita(27), yolk and white together, Stepanekto)) and alcohol (yolk and white together, Stepanek(2s)).
Arising out of these points we had the following considerations:
(1) True fermentation, the production of alcohol as well as lactic acid from glucose, occurs nowhere in the animal kingdom. Can the earlier workers have been right in observing it in hen's eggs? Were their experiments conducted with really stringent bacteriological sterility?
(2) What part does the vitelline membrane play in the lactic acid production? Is it capable of bringing about glycolysis in the absence of yolk, and if so, is it responsible for all the glycolysis of the infertile egg?
(3) Does the vitelline membrane or the yolk respire in vitro}
(4) Does the whole infertile egg show any true respiration? This could only be answered by making a fresh study of the carbon dioxide production and com-paring it with the oxygen consumption.
(5) Does the vitelline membrane show any catalytic activity with glucose or other hydrogen donators and methylene blue in the Thunberg tube?
(6) If a vitelline membrane were placed in a specially constructed dialysing apparatus, with a solution of salts equivalent to the yolk on one side, and a solution of salts equivalent to the white on the other side, would it maintain the difference or come quickly to equilibrium?
(7) Similarly, what would be the behaviour of intact yolks suspended in hypertonic and hypotonic solutions of electrolytes and non-electrolytes ?
(8) At room temperature the freezing-points of yolk and white have approached each other but have not become identical after two months. How is the equilibra-tion achieved, and how does the process depend on temperature?
(9) Does viability bear any relation to the decline of difference in freezing-point between yolk and white? It is known that viability of fertile eggs declines in a regular curve and has an optimum temperature (Moranos)), and it is known that
viability, like osmotic pressure difference, may be retained for some time in atmospheres of hydrogen (AnceltO, Lallemandd3)).
(10) What evolutionary significance has the difference in osmotic pressure between yolk and white, and at what point in the evolution of the terrestrial egg did it begin to acquire its importance ?
The succeeding papers of this series give the answers to these questions, in so far as we have been able to find them.
REFERENCES.
(1) ANCEL, S. (1928, 1929). Arch. d'Anat. d'Histol. et d'Embryol. 8, 434; 9, 1. (2) AOKI, M. (1925). Japanese Journ. Biochem. 5, 71.
(3) BALFOUR, F. M. (1887) Comparative Embryology.
(4) BIALASCEWICZ, K. (1912). Arch.f.Enttvicklungsmech.3i,^8<).
(5) BIALASCEWICZ, K. and BLEDOVSKI, R. (1915). Proc. Sci. Soc. Warsaw, 9, 429.
(6) CHICK, H., COPPING, A. M. and ROSCOE, M. H. (1931). Biochem. Journ. 24, 1748.
(7) GRAY, J. Private communication to the authors. (8) HAYES, F. R. (1930). Zeitschr. f. vergl. Physiol. 13, 214. (9) HILL, A. V. (1930). Proc. Faraday Soc. (Symposium). (10) (1930), Proc. Roy. Soc. B, 106, 477.
(11) IDO, R. (1930). Arb. a. d. Med. Univ. Okayama, 2, 127. (12) KAMEI, K. (1929). Zeitschr. f. physiol. Chem. 181, 101. (13) LALLEMAND, S. (1929). Journ. Pharm. Chim. Ser. 8, 9, 380.
(14) LANGWORTHY, C. F . and BAROTT, H. G. (1921). Journ. Biol. Chem. 46, xlix. (15) LECAILLON, A. (1910). C.R. Acad. Sci. 150, 240; C.R. Soc. Biol. 68, 218. (16) LIEBERMANN, L. v. (1888). Arch.f. d. g. Physiol. 43, 71.
(17) MARGARIA, R. (1930). Journ. Physiol. 70, 417. (18) MORAN, T . (1925). Proc. Roy. Soc. B, 98, 436.
(19) NASSONOV, D. (1930). Zeitschr. f. Zellforsch. u. mik. Anat. 11, 179. (20) OSBORNE, W. A. and KINCAID, H. E. (1914). Biochem. Journ. 8, 28. (21) PANTIN, C. F. A. Private communication to the authors.
