The Organism as a Whole, from a Physicochemical Viewpoint

But how can membrane formation be produced by mere agitation? It seems to me that this can be understood if we suppose that it depends upon the destruction of an emulsion in the cortical layer of the egg. It is conceivable that in the egg of certain forms the stability of this emulsion is so small that mere shaking would be enough to destroy it and thus induce membrane formation and development.1 The durability of emulsions varies, and where an emulsion is very durable shaking has no effect, while where it is at the critical point of separating into two continuous phases a slight shaking will bring about the separation, and where the emulsion is still less durable we observe the phenomenon of a "spontaneous" parthenogenesis. Eggs like those of most sea urchins belong to the former, eggs like those of some starfish and annelids belong to the second or third type.

1 Loeb, J., Artificial Parthenogenesis and Fertilization, p. 255. ization membrane is preformed in the fertilized egg and merely lifted off from the egg or whether its formation is due to the hardening of a colloidal substance separated from the emulsion (or excreted) and hardened in touch with sea water. But we can be sure of one thing, namely, that the liquid between egg and membrane contains some colloidal substance which determines the tension and spherical shape of the membrane. The membrane is obviously permeable not only to water but also to dissolved crystalloids, while it is impermeable to colloids. When we add some colloidal solution (e. g., white of egg, blood serum, or tannic acid) to the sea water containing fertilized eggs of purpuratus, the membrane collapses and lies close around the egg; while if the eggs are put back into sea water or a sugar solution the membrane soon assumes its spherical shape. This is intelligible on the assumption that in the process of membrane formation (or in the destruction of the emulsion in the cortical layer) a colloidal substance goes into solution which cannot diffuse into the sea water since the membrane is impermeable to the colloidal particles. The membrane is, however, permeable to the constituents of sea water or to sugar. Consequently sea water will diffuse into the space between membrane and egg until the tension of the membrane equals the osmotic pressure of the colloid dissolved in the space between egg and the membrane. If we add enough colloid to the outside solution so that

its osmotic pressure is higher than that of the colloidal solution inside the membrane the latter will collapse. It should also be stated that the unfertilized eggs of many marine animals are surrounded by a jelly (chorion) which is dissolved when the egg is fertilized. * The writer has shown that the same chemical substances which will induce membrane formation and artificial parthenogenesis will as a rule also cause a swelling and liquefaction of the chorion.

We have devoted so much space to the mechanism of membrane formation since it is likely to give a clearer insight into the physicochemical nature of physiological processes than the phenomena of muscular stimulation and contraction or nerve stimulation, upon which the majority of physiologists base their conclusions concerning the mechanism of life phenomena. Before we come to the discussion of the second factor in the activation of the egg it should be stated more definitely that for the eggs of some forms the first factor, the process underlying membrane formation, suffices for the development of the egg into a larva and that no second factor is required in these cases. This is true for the eggs of starfish and certain annelids.

1 It has been stated by several writers that the eggs of the sea urchin can no longer form the fertilization membrane when the jelly surrounding the egg is dissolved. The writer has found that if the jelly surrounding the eggs of Stronvylocentrotus purpuratus is dissolved by acid the eggs still form a fertilization membrane upon the entrance of a spermatozoon. Thus in 1901 Loeb1 and Neilson showed that a short treatment with HC1 and HNO3 sufficed to cause some eggs of Asterias in Woods Hole to develop into larvae without a second treatment being needed, and Delage2 showed the same for C02; and in 1905 the writer found that the eggs of the Calif ornian starfish Asterina can be induced to form a membrane by butyric acid treatment and that ten per cent, of these eggs developed into normal larvae. Quite recently R. S. Lillie observed that the eggs of Asterias at Woods Hole can be caused to form membranes and develop into larvae by a treatment with butyric acid and that the time of exposure required to get a maximal number of larvae varies approximately inversely with the concentration of the acid, within a range of 0.0005 to 0.006 N butyric acid. If the exposure is too short membrane formation will occur without normal development. 3

All this leads us to the conclusion that the main effect of the spermatozoon in inducing the development of the egg consists in an alteration of the surface of the latter which is apparently of the nature of a cytolysis of the cortical layer. Anything that causes this alteration without endangering the rest of the egg may induce its development. The spermatozoon, therefore, causes the development of the egg by carrying a substance into the latter which effects an alteration of its surface layer.

5. We will now discuss the action of the second, corrective factor, in the inducement of development. When we cause membrane formation in a sea-urchin eS2 by the proper treatment with butyric acid it will commence to develop and segment but will disintegrate rapidly if kept at room temperature and the more rapidly the higher the temperature. If, however, the eggs are treated afterward for a certain length of time (from thirty-five to sixty minutes at 15° C. for purpuratus and 17^2 to 22^2 minutes for Arbacia at 23° C.) in a solution which is isosmotic with 50 c.c. sea water +8 c.c. 2^/2 m NaCl, r they will develop into larvae, many of which may be normal. Any hypertonic solution of this osmotic pressure, sea water, sugar, or a single salt, will suffice provided the solution does not contain substances that are too destructive for living matter. The hypertonic solution produces its corrective effect only if the egg contains free oxygen; and in a slightly alkaline medium more rapidly than in a neutral medium. The time of exposure in the hypertonic solution dimin-

1 It is necessary to call attention to the fact that sugar solutions of a high concentration (e. g.t m solutions) have a much higher osmotic pressure than that which they should have theoretically (Lord Berkeley and Hartley). Delage by ignoring this fact has misinterpreted his experiments with sugar solutions. See Lloyd, D. J., Arch.f. Entwcklngsmech., 1914, xxxviii., 402. ishes in certain limits with the concentration of OH ions in the solution.

It is strange that in the eggs of purpuratus the corrective effect can also be brought about by exposing the eggs after the artificial membrane formation for about three hours to normal sea water free from oxygen ; or to sea water in which the oxidations have been retarded by the addition of KCN. This method is not so reliable as the treatment with hypertonic solution. What does the hypertonic solution do to prevent the disintegration of the egg after the artificial membrane formation? The writer suggested in 1905 that the artificial membrane formation alone starts the development but leaves the eggs usually in a sickly condition and that the hypertonic solution or the lack of oxygen allows them to recuperate from such a condition. The second factor is, according to this view, merely a corrective or curative factor. The following observations will explain the reasons for such an assumption.

The writer found that if we keep the unfertilized eggs after artificial membrane formation in sea water deprived of oxygen the disintegration of the egg following artificial membrane formation is prevented for a day at least. The same result can be obtained by adding ten drops of A, per cent. KCN to 50 c.c. of sea water, and certain narcotics, e. g., chloral hydrate, act in the same way. Wasteneys and the writer found that chloral hydrate (and other narcotics) in the con-

centration required do not suppress or even lower the oxidations in the egg to any considerable extent,1 but they prevent the processes of cell division. Hence it seems that the egg disintegrates so rapidly after artificial membrane formation because it is killed by those processes leading to nuclear division or cell division which are induced by the artificial membrane formation. If we suppress these phenomena of development (for not too long a time) we give the egg a chance to recover and if now the impulse to develop is still active we notice a perfectly normal development. If the egg is kept too long without oxygen it suffers for other reasons and cannot develop ; the writer has shown that if eggs fertilized by sperm are kept for too long a time without oxygen they also will no longer be able to develop normally. The short treatment with a hypertonic solution supplies the corrective factor required, so that the egg can then undergo cell division at room temperature without disintegrating.

The correctness of this interpretation, which is in reality mainly a statement of observations, is proved by the two following groups of facts. The older observers had already noticed that the unfertilized eggs of the sea urchin when lying in sea water will die after a day or more, and that occasionally such eggs show nuclear division or even the beginning of cell division shortly before disintegration sets in. The writer has studied this phenomenon in the unfertilized eggs of purpuratus and found that only the eggs of certain females show this cell division before disintegration and that the cell division is preceded by an atypical form of membrane formation; the eggs surrounding themselves by a fine gelatinous film comparable to that produced in the egg of Arbacia by a treatment with butyric acid. It is difficult to state what induces the alteration of the surface in the eggs that lie so long in sea water. It may be due to the CO2 formed by the eggs — since we know that C02 may induce membrane formation — or it may be due to the alkalinity of the sea water or to a substance originating from the jelly surrounding the eggs. It was found that if such eggs are kept without oxygen their disintegration (and cell division) will be delayed considerably. The presumable explanation for this is that the lack of oxygen prevents the internal changes underlying cell division and thus prevents the disintegration of the egg. The direct proof that an egg in the process of cell division is more endangered by abnormal solutions than an egg at rest has been furnished by numerous observations of the writer. He showed in 1906 that the fertilized egg of purpuratus dies rather rapidly in a pure m/2 NaCl or any other abnormal isotonic solution, while the unfertilized egg can live for days in such solutions.1 In

a series of papers, beginning in 1905, he showed that the fertilized egg will live longer in hypertonic, hypotonic, and otherwise abnormally constituted solutions when the cell divisions are suppressed by lack of oxygen or by the addition of KCN or of chloral hydrate.1 It is thus obvious that coincident with the changes underlying nuclear division or cell division alterations occur in the sensitiveness of the egg to salt solutions of abnormal concentration or constitution, e. g.t NaCl+CaCla isotonic with sea water, hypertonic, or hypotonic solutions.

We must, therefore, conclude that artificial membrane formation induces development but that it leaves the egg in a sickly condition in which the very processes leading to cell division bring about its destruction; that if it is given time it can recover from this condition and that the treatment with the hypertonic solution also brings about this recovery rapidly and reliably. Herlant2 suggested that the corrective effect of the hypertonic solution consisted in the proper development of the astrospheres required for cell division. According to this author mere membrane formation does not lead to the formation of sufficiently large astrospheres and hence cell division may remain im-

possible. x The writer has no a priori objection to this suggestion which agrees with earlier observations by Morgan except that it is at present difficult to harmonize it with all the facts.' Why should it be possible to replace the treatment with the hypertonic solution by a suspension of the oxidations in the egg for three hours while we know that lack of oxygen suppresses the formation of astrospheres in the fertilized eggs? What becomes of the astrospheres if the treatment with the hypertonic solution precedes the membrane formation by a number of hours or a day (which is possible as wTe shall see), and why do they not induce cell division, if Herlant's idea is correct? Nevertheless the suggestion of Herlant deserves to be taken into serious consideration.

6. How can an alteration of the surface of the egg — e. g., a cytolytic or other destruction of the cortical layer — lead to a beginning of development? The answer is possibly given in the relation of oxidation to development. The writer found in 1895 that if oxygen is withdrawn from the fertilized sea-urchin egg it can not segment and this seems to be the case for eggs in general.2 In 1906 he found that the rapid disintegration of the eggs of the sea urchin which follows artificial

1 It is also important to remember that the formation of astrospheres after mere membrane formation occurs considerably more slowly than if the egg has also received a treatment with a hypertonic solution. 2 The writer found that the eggs of Fundulus will segment a number of times even if all the oxygen has apparently been removed. membrane formation could be prevented when the eggs were deprived of oxygen or when the oxidations were suppressed in the eggs by KCN. This suggested a connection between the disintegration of the egg after artificial membrane formation and the increase in the rate of oxidations ; and he found further that the formation of acid is greater in the fertilized than in the unfertilized egg. He, therefore, expressed the view in 1906 that the essential feature (or possibly one of the essential features) of the process of fertilization was the increase of the rate of oxidations in the egg and that this increase was caused by the membrane formation alone. x These conclusions have been since amply confirmed by the measurements of O. Warburg as well as those of Loeb and Wasteneys, both showing that the entrance of the spermatozoon into the egg raises the rate of oxidations from 400 to 600 per cent., and that membrane formation alone brings about an increase of similar magnitude. Loeb and Wasteneys found that the hypertonic solution does not increase the rate of oxidations in a fertilized egg. It does do so, however, in an unfertilized egg without membrane formation, but merely for the reason that in such an egg the hypertonic solution brings about the cytolytic change in the cortex of the egg underlying membrane formation. a

2 Thus the treatment of an unfertilized egg without membrane with a hypertonic solution combines two effects, first the general cytolytic According to Warburg it is probable that the oxidations occur mainly if not exclusively at the surface of the egg since NaOH, which does not diffuse into the egg, raises the rate of oxidations more than NH4OH which does diffuse into the egg. And finally, the same author showed that the oxidations in the sea-urchin egg are due to a catalytic process in which iron acts as a catalyzer.1 In view of all these facts and their harmony with the methods of artificial parthenogenesis the suggestion is justifiable that the alteration or cytolysis of the cortical layer of the egg is in some way connected with the increased rate of oxidations. The question remains then : How can membrane formation or the alteration of the cortical layer underlying membrane formation cause an increase in the rate of oxidations? One possibility is that the iron (or whatever the nature of the catalyzer may be) exists in the cortex of the egg in a masked condition — or in a condition in which it is not able to act — while the alteration of the cortical "layer makes the iron active. It might be that either the iron or the oxidizable substrate is contained in the lipoid layer in the unfertilized condition of the egg and that the destruction or cytolysis of the cortical layer brings both the iron and the oxidizable substrate into the watery phase in which they can interact.

alteration of the cortical layer of the membrane and the corrective effect of the hypertonic solution. The former effect raises the rate of oxidations in the egg, the latter does not. Another possibility is that the act of fertilization increases the permeability of the egg. This idea, which seems attractive, was first suggested and discussed by the writer in 1906.' He had found that when fertilized and unfertilized eggs were put into abnormal salt solutions, e. g.t pure solutions of NaCl, the fertilized eggs died more rapidly than the unfertilized eggs and he pointed out that these experiments suggested the possibility that fertilization increases the permeability of the egg for salts. The reason for his hesitation to accept this interpretation was, that the fertilized egg is also more easily injured by lack of oxygen than the unfertilized egg and in this case the greater sensitiveness of the fertilized egg was obviously due to its greater rate of metabolism. Later experiments by the writer showed that the fertilized egg can be made more resistant to abnormal salt solutions if its development is suppressed by lack of oxygen or by KCN or by certain narcotics. With our present knowledge it does not seem very probable that lack of oxygen diminishes the permeability of the egg, but we know that it inhibits the developmental processes. Warburg has made it appear very probable that the fertilized egg is impermeable for NaOH and if this is the case it should also be impermeable for NaCl. 2

2 Unless the egg is left so long in the pure NaCl solution that its permeability is increased. The idea that fertilization and membrane formation cause an increase in the permeability of the egg was later accepted and elaborated by R. Lillie. This author assumes that the unfertilized egg cannot develop because it contains too much C02 but that the CO 2 can escape from the egg as soon as its permeability is increased through the destruction of the cortical layer of the egg.1 After the C02 has escaped, the excessive permeability must be restored to its normal value and this is the r61e of the hypertonic treatment. It is, however, difficult to harmonize the assumption of an impermeability of the unfertilized egg for CO2 with the fact that if the unfertilized sea-urchin egg is cut into two, as is done in merogony, no development takes place, while such pieces will develop when a spermatozoon enters. The cortical layer is removed along the cut surface and there is no reason why the CO 2 should not escape. Besides, the experiments of Godlewski and the writer prove that the cortical layer of the unfertilized sea-urchin egg is apparently very permeable for C02 since the latter causes membrane formation if contained in the sea water in sufficiently high concentration.

Lillie assumes that the hypertonic treatment restores the permeability raised to excess by the butyric acid treatment, but this assumption is not in harmony with the following facts. The writer has shown that it is immaterial whether the eggs are treated first with the hypertonic solution and then with butyric acid or the reverse, if only the eggs remain longer in the hypertonic solution when the hypertonic treatment precedes the butyric acid treatment. It was stated in the beginning of this chapter that the development of the egg can be induced by hypertonic sea water, and we know the reason since hypertonic sea water is a cytolytic agency. The writer found that when we expose unfertilized eggs of purpuratus for from two to two and a halfhours to hypertonic sea water they will often not develop and only a few eggs will undergo the first cell divisions, then going into a condition of rest. Y/hen these eggs, both the segmented and unsegmented, were treated twenty-four or thirty-six hours later with butyric acid, so that they formed a membrane, they all developed into larvae without further treatment. It is impossible to apply Lillie's theory to these facts, for the simple reason that the treatment with hypertonic sea water was just long enough to induce development in some eggs and hence according to Lillie's ideas must have increased the permeability of these eggs. Yet these same eggs were induced to develop normally when subsequently treated with butyric acid, which according to Lillie also acts by increasing the permeability. Nothing indicates that the treatment of the eggs with a hypertonic solution diminishes

their permeability; the reverse would be much more probable. Lillie's theory also fails to explain that mere treatment of the eggs with a hypertonic solution can bring about their development into larvae. This, however, is intelligible on the assumption that the hypertonic solution in this case has two different effects, first a cytolysis of the cortical layer of the egg and second an entirely different effect, possibly upon the interior of the egg, which represents the second or corrective effect.

McClendon1 has shown that the electrical conductivity of the egg is increased after fertilization, and J. Gray2 has found that this increase in conductivity is only transitory and disappears in fifteen minutes. This might indicate that the egg becomes transitorily more permeable for salts after the entrance of the spermatozoon or after membrane formation; although an increase in conductivity might be caused by other changes than a mere increase in permeability of the egg. The writer is of the opinion that it is necessary to meet all these and other difficulties before we can state that the alteration of the cortical layer, which is the essential feature of development, acts chiefly or exclusively by an increase in the permeability of the egg. 3

3 R. Lillie has recently shown that in a hypotonic solution water diffuses more rapidly into a fertilized than into an unfertilized egg. 7. When the experiments on artificial parthenogenesis were first published they aroused a good deal of antagonism not only among reactionaries in general but also among a certain group of biologists. O. Hertwig had defined fertilization as consisting in the fusion of two nuclei, the egg nucleus and the sperm nucleus. No such fusion of two nuclei takes place in artificial parthenogenesis since no spermatozoon enters the egg, and it became necessary, therefore, to abandon Hertwig's definition as wrong. The objection raised that the phenomena are limited to a few species soon became untenable since it has been possible to produce artificial parthenogenesis in the egg of plants (Fucus, according to Overton) as well as of animals, from echinoderms up to the frog; and it may possibly one day be accomplished also in warm-blooded animals. A second objection was that the eggs caused to develop by the methods of artificial parthenogenesis could never reach the adult stage and that hence the phenomenon was merely pathological. There was no basis for such a statement, except that it is extremely difficult to raise marine invertebrates. Delage1 was courageous enough

This is exactly what one should expect since the unfertilized egg is not only surrounded by the cortical layer but also by a thick layer of jelly both of which are lacking in the fertilized egg. It is difficult to understand how this observation can throw any light on the mechanism of development, since water diffuses rapidly enough into the unfertilized egg. to make an attempt to raise parthenogenetic larvae of the sea urchin beyond the larval stage and he succeeded in one case in carrying the animal to the mature stage. It proved to be a male.

Better opportunities were offered when a method was discovered which induced the development of the unfertilized eggs of the frog. In 1907, Guyer made the surprising observation that if he injected lymph or blood into the unfertilized eggs of frogs he succeeded in starting development and he even obtained two free-swimming tadpoles. ' ' Apparently the white rather than the red corpuscles are the stimulating agents which bring about development, because injections of lymph which contains only white corpuscles produce the same effects as injections of blood." Curiously enough, Guyer thought that probably the cells which he introduced and not the egg were developing. In 1910, Bataillon showed that a mere puncture of the egg with a needle could induce development but he believes that for the full development the introduction of a fragment of a leucocyte is required. Bataillon has called attention to the analogy with the writer's results on lower forms, the puncturing of the egg corresponding to the cytolysis of the surface layer of the egg and the introduction of a leucocyte as the analogue of the second or corrective factor. The method of producing artificial parthenogenesis by puncturing the egg has thus far been successful only in the egg of

the frog. The writer has tried it in vain on the eggs of many other forms. He has at present seven parthenogenetic frogs over a year old, produced by merely puncturing the eggs with a fine needle (Fig. 6). These frogs have reached over half the size of the adult frog. They can in no way be distinguished from the frogs produced by fertilization with a spermatozoon. This makes the proof conclusive that the methods of artificial parthenogenesis can result in the production of normal organisms which can reach the adult stage.

Bancroft and the writer tried to determine the sex of a parthenogenetic tadpole and of a frog just carried through metamorphosis. Since in early life the sex glands of both sexes in the frog contain eggs it is not quite easy to determine the sex, except that in the male the eggs gradually disappear and from this and other criteria we came to the conclusion that both parthenogenetic specimens, which were four months old, were males. The writer has recently examined the gonads of a ten months old parthenogenetic frog. Here no doubt concerning the sex was possible since the gonads were well-developed testicles containing a large number of spermatozoa of normal appearance, and no eggs. x (Figs. 7 and 8.) This would indicate that the frog belongs to those animals in which the male is heterozygous for sex.

1 Since this was written, two more of the parthenogenetic frogs over a year old died. Both were males. 8. The fact that the egg of so high a form as the frog can be made to develop into a perfect and normal animal without a spermatozoon — although normally the egg of this form does not develop unless a spermatozoon enters — corroborates the idea expressed in previous chapters that the egg is the future embryo and animal; and that the spermatozoon, aside from its activating effect, only transmits Mendelian characters to the egg. The question arises : Is it possible to cause a spermatozoon to develop into an embryo? The idea has been expressed that the egg was only the nutritive medium on which the spermatozoon developed into an embryo, but this idea has been rendered untenable by the experiments on artificial parthenogenesis. Nevertheless the question whether or not the spermatozoon can develop into an embryo on a suitable culture medium remains, and it can only be decided by direct experiments. It was shown by Boveri, Morgan, Delage, Godlewski, and others, that if a spermatozoon enters an enucleated egg or piece of egg it can develop into an embryo, but since the cytoplasm of the egg is the future embryo this experiment proves only that the egg nucleus may be replaced by the sperm nucleus; and also that the sperm nucleus carries into the egg the substances which induce development. Ineidentally these experiments on merogony also prove that the mere mechanical tearing of the cortical layer,— which must happen in the separation of the unfertilized

egg into parts with and without a nucleus, — by dissection or by shaking, is not sufficient to start development in the sea-urchin egg. J. de Meyer put the spermatozoa of sea urchins into sea water containing an extract of the eggs of the same species but found only that the spermatozoa swell in such a solution. Loeb and Bancroft made extensive experiments in cultivating spermatozoa of fowl in vitro on suitable culture media. In yolk and white of egg the head of the spermatozoon underwent transformation into a nucleus, but no mitosis or aster formation was observed.1 These experiments should be continued.