ARTIFICIAL PARTHENOGENESIS

1. The majority of eggs cannot develop unless they are fertilized, that is to say, unless a spermato­zoön enters into the egg. The ques­tion arises: How does the spermato­zoön cause the egg to develop into a new organism? The spermato­zoön is a living organism with a complicated structure and it is impossible to explain the causa­tion of the development of the egg from the structure of the spermato­zoön. No progress was possible in this field until ways were found to replace the action of the living spermato­zoön by well-known physico­chemical agencies.[84] Various observers such as Tichomiroff, R. Hertwig, and T. H. Morgan had found that unfertilized eggs may begin to segment under certain condi­tions, but such eggs always disintegrated in their experi­ments without giving rise to larvæ. In 1899 the writer succeeded in causing the unfertilized eggs of the sea urchin Arbacia to develop into swimming larvæ, blastulæ, gastrulæ, and plutei, by treating them with hypertonic sea water of a definite osmotic pressure for about two hours. When such eggs were then put back into normal sea water many segmented and a certain percentage developed into perfectly normal larvæ, blastulæ, gastrulæ, and plutei.[85] Soon afterward this was accomplished by other methods for the unfertilized eggs of a large number of marine animals, such as starfish, molluscs, and annelids. None of these eggs can develop under normal condi­tions unless a spermato­zoön enters. These experi­ments furnished proof that the activating effect of the spermato­zoön upon the egg can be replaced by a purely physico­chemical agency.[86]

The first method used in the produc­tion of larvæ from the unfertilized eggs did not lend itself to an analysis of the activating effect of the spermato­zoön upon the egg, since nothing was known about the action of a hypertonic solu­tion, except that it withdraws water from the egg; and there was no indica­tion that the entrance of the spermato­zoön causes the egg to lose water. No further progress was possible until another method of artificial parthenogenesis was found. When a spermato­zoön enters the egg of a sea urchin or starfish or certain annelids, the surface of the egg undergoes a change which is called membrane forma­tion; and which consists in the appearance of a fine membrane around the egg, separated from the latter by a liquid (Figs. 4 and 5). O. and R. Hertwig and Herbst had observed that such a membrane could be produced in an unfertilized egg if the latter was put into chloroform or xylol, but such eggs perished at once. It was generally assumed, moreover, that the process of membrane forma­tion was of no significance in the phenomenon of fertiliza­tion, except perhaps that the fertiliza­tion membrane guarded the fertilized egg against a further invasion by sperm. However, since the fertilized egg is protected against this possibility by other means the membrane is hardly needed for such a purpose.

Fig. 4    Fig. 5
Fig. 4. Unfertilized egg surrounded byspermatozoa (whose fla­gel­lum is omitted in the drawing).
Fig. 5. The same egg after a spermato­zoönhas entered. The fer­til­iza­­tion membrane is separated from the egg by aclear space.

In 1905 the writer found that membrane forma­tion, or rather the change of the surface of the egg underlying the membrane forma­tion, is the essential feature in the activa­tion of the egg by a spermato­zoön. He observed that when unfertilized eggs of the Californian sea urchin Strongylo­centrotus purpuratus are put for from one and a half to three minutes into a mixture of 50 c.c. of sea water+2.6 c.c. N/10 acetic or propionic or butyric or valerianic acid and are then put into normal sea water all or the majority of the eggs form membranes; and that such eggs when the temperature is very low will segment once or repeatedly and may even—if the temperature is as low as 4°C. or less—develop into swimming blastulæ[87]; but they will then disintegrate. On the other hand, if they are kept at room temperature they will develop only as far as the aster forma­tion and nuclear division and then begin to disintegrate. It should be men­tioned that the time which elapses between artificial membrane forma­tion and nuclear division is greater than that between the entrance of a spermato­zoön and nuclear division.

It was obvious, therefore, that artificial membrane forma­tion induced by butyric acid initiates the processes underlying development of the egg but that for some reason the egg is sickly and perishes rapidly.

When, however, such eggs were given a short treatment with hypertonic sea water or with lack of oxygen or with KCN they developed into normal larvæ. This new or improved method of artificial parthenogenesis is as follows: The eggs are put for from two to four minutes into 50 c.c. sea water containing a certain amount of N/10 butyric acid (2.6 c.c. in the case of S. purpuratus in California and 2.0 c.c. in the case of Arbacia in Woods Hole). Ten or fifteen minutes later the eggs are put into hypertonic sea water (50 c.c. sea water+8 c.c. 21⁄2 m NaCl or Ringer solu­tion or cane sugar) in which they remain, at 15° C. from thirty-five to sixty minutes in the case of purpuratus, and from 171⁄2 minutes to 221⁄2 minutes at 23° in the case of Arbacia at Woods Hole. If the eggs are then transferred to normal sea water they will develop. In making these experi­ments, which have been repeated and confirmed by numerous investigators, it should be remembered that this effect of the hypertonic solu­tion has a high temperature coefficient (about two for 10° C.) and that a slight overexposure to the hypertonic sea water injures the eggs so that development is abnormal. By this method it is possible to imitate the activating effect of the living spermato­zoön upon the egg in every detail and eggs treated in this way will develop in large numbers into perfectly normal larvæ. We shall see later that they can also be raised to the adult state.

2. The next task was to find out the nature of the action of the two agencies upon the development of the egg. It soon became obvious that the membrane forma­tion (or the altera­tion underlying membrane forma­tion) was the more important of the two, since in the eggs of starfish and annelids this was sufficient for the produc­tion of larvæ, and that the second treatment had only the corrective effect, of overcoming the sickly condi­tion in which mere membrane forma­tion had left the eggs. It was, therefore, of great interest to ascertain what substances or agencies caused membrane forma­tion in the egg, since it now became clear that the spermato­zoön could only cause membrane forma­tion by carrying one such substance into the egg. These investiga­tions led the writer to the result that all those substances and agencies which are known to cause cytolysis or hemolysis (see Chapter III) will also induce membrane forma­tion, and that the essential feature in the causa­tion of development is a cytolysis of the superficial or cortical layer of the egg. As soon as this layer is destroyed the development of the egg can begin.

The substances and agencies which cause cytolysis and hence, if their action is restricted to the surface of the egg, will induce development are, besides the fatty acids: (1) saponin or solanin or bile salts; (2) the solvents of lipoids, benzol, toluol, amylene, chloroform, aldehyde, ether, alcohols, etc.; (3) bases; (4) hypertonic or hypotonic solu­tions; (5) rise in temperature, and (6) certain salts, e. g., BaCl2 and SrCl2 in the case of the egg of purpuratus, and according to R. Lillie, NaI or NaCNS in the egg of Arbacia. Whenever we submit an unfertilized sea-urchin egg to any of these agencies and restrict the cytolysis to the superficial or cortical layer of the egg (i. e., if we transfer the egg to normal sea water before the cytolytic agent has had time to diffuse into the main egg) the egg will form a membrane and behave as if the membrane forma­tion had been called forth by a fatty acid, with this difference only, that the various agencies are not all equally harmless for the egg.[88]

If the idea was correct that the change underlying membrane forma­tion was essentially a cytolysis of the cortical layer of the egg, it was to be expected (from the data contained in Chapter III) that the blood serum or the cell extracts of foreign species would also cause membrane forma­tion and thus induce the development of the unfertilized egg, while serum of animals of the same species or genus would have no such effects. This was found to be correct. In 1907 the writer showed that the blood serum of a Gephyrean worm, Dendrostoma, was able to cause membrane forma­tion in the egg of the sea urchin. When added in a dilu­tion of 1 c.c. of serum to 500 or 1000 c.c. of sea water to eggs of purpuratus a certain number formed fertiliza­tion membranes. It was found later that the serum and tissue extracts of a large number of animals, especially of mammals (rabbit, pig, ox, etc.), had the same effect, though it was necessary to use higher concentra­tions, one-half sea water and one-half isotonic blood serum. The eggs of every female sea urchin, however, did not give the reac­tion and not all the eggs even of sensitive females formed membranes. The writer found, however, that it was possible to increase the susceptibility of the eggs against foreign blood serum by putting them into a 3⁄8 m solu­tion of SrCl2 for from five to ten minutes (or possibly a little longer) before exposing them to the foreign blood serum. BaCl2 acts similarly. The fact that SrCl2 alone can cause membrane forma­tion in unfertilized eggs if they are left long enough in the solu­tion suggests that the sensitizing effect of the substance consists in a modifica­tion of the cortical layer similar to that underlying membrane forma­tion; and that the subliminal effect of a short treatment with SrCl2 and the subliminal effect of the foreign serum when combined suffice to bring about the membrane forma­tion.

Not only the watery extract of foreign cells but also that of foreign sperm, induces membrane forma­tion in the sea-urchin egg. The watery extract of sperm of starfish is especially active, but the degree of activity varies considerably with the species of starfish from which the sperm is taken. The eggs of different species of sea urchins also show a different degree of susceptibility for the sperm of foreign species. Thus the eggs of Strongylo­centrotus purpuratus require a higher concentra­tion of sperm extract than the eggs of S. franciscanus. For the latter the amount of foreign cell constituents which suffices to call forth membrane forma­tion is so small that contact with almost any foreign living spermato­zoön produces this effect; and as a rule no previous sensitizing action of SrCl2 is required. When we bring the unfertilized eggs of S. franciscanus into contact with the living sperm of starfish or shark or even of fowl, the eggs form a fertiliza­tion membrane without previous sensitiza­tion. A specific substance from the foreign spermato­zoön causes membrane forma­tion before the spermato­zoön has time to enter the egg. The effect is the same as if artificial membrane forma­tion had been called forth with butyric acid, i. e., they begin to develop and then disintegrate unless they receive a second short treatment.

When, however, we treat the eggs with the watery extracts from the cells of their own or closely related species we find that these extracts are utterly inactive, even if used in comparatively strong concentra­tions. This agrees with the results given in Chapter III.

These phenomena lead to a very paradoxical result; namely that while in the case of foreign sperm we can cause membrane forma­tion by both the living and the dead spermato­zoön, only the living spermato­zoön of the same species can induce membrane forma­tion. This might find its explana­tion on the assump­tion that the active substance contained in the foreign sperm or serum is water-soluble and a protein, while the activating or membrane-forming substance in the spermato­zoön is insoluble in water but soluble in the egg (or in lipoids). If this assump­tion is correct the two substances are essentially different.

Robertson[89] has succeeded in extracting a substance from the sperm of the sea urchin which causes membrane forma­tion of the sea-urchin egg after the latter has been sensitized by a treatment with SrCl2. It seems to the writer that if the substance extracted by Robertson were the real fertilizing agent contained in the spermato­zoön it should fertilize the egg without a previous sensitiza­tion of the egg with SrCl2 being required.

3. The action of acids in the mechanism of artificial parthenogenesis provides some interesting physio­logical problems. When unfertilized sea-urchin eggs are left in sea water containing any of the lower fatty acids up to capronic, the eggs will form no membranes, while in such sea water, and they will show no outer signs of cytolysis (swelling). When, however, the eggs are left in sea water containing any of the fatty acids from heptylic upward the eggs will form membranes while in the acid sea water and soon afterward will cytolyze completely and swell enormously. In solu­tions of the mineral acids no membranes are formed and none are formed as a rule when the eggs are transferred back to sea water. When both a mineral and a lower fatty acid, e. g., butyric, are added to sea water the mineral acid acts as if it were not present, i. e., the eggs form membranes when transferred back to sea water if the concentra­tion of the butyric acid is high enough. All these data are comprehensible if we assume that only that part of the acid causes membrane forma­tion which is lipoid soluble, while the water soluble part is not involved in the process of membrane forma­tion; and that the cytolysis or swelling of the whole egg can only take place in the higher fatty acids (heptylic or above) which are little soluble in water and very soluble in lipoids, while the lower fatty acids, whose water solubility is comparatively high, can only bring about a cytolysis and swelling in the cortical layer but not in the rest of the egg. This makes it appear as though the part undergoing an altera­tion in membrane forma­tion was a lipoid; and this would harmonize with the assump­tion that the specific membrane-inducing substance in the spermato­zoön is not soluble in water, but soluble in fat.

4. These and other observa­tions led the writer to the view that the essential process which causes development might be an altera­tion of the surface of the egg, in all probability an altera­tion of the superficial layer probably of the nature of a superficial cytolysis. The ques­tion remains: What could be the physico­chemical nature of this cytolysis? The writer had suggested in former papers that in the cytolysis underlying membrane forma­tion lipoids were dissolved, and he supposed that the substance to be dissolved might be a calcium-lipoid compound which might form a continuous layer under the surface of the egg.[90] v. Knaffl, working on the cytolysis of eggs in the writer’s laboratory, gave the following idea of the process:

Protoplasm is rich in lipoids; probably it is mainly an emulsion of these and proteins. Any physical or chemical stimulus which can liquefy the lipoids causes cytolysis of the egg. The protein of the egg can really only swell or be dissolved if the condi­tion of aggrega­tion of the lipoid is altered by chemical or physical agencies. The mechanism of cytolysis consists in the liquefac­tion of the lipoids and thereupon the lipoid-free protein swells or is dissolved by taking up water. . . . Hence this supports Loeb’s view that membrane forma­tion is induced by the liquefac­tion of lipoids.[91]

The writer suggested that the destruc­tion of an emulsion in the cortical layer might possibly be the essential feature of the altera­tion leading to membrane forma­tion and development. It had been long observed that unfertilized starfish eggs may begin to develop apparently without any outside “stimulus,” and A. P. Mathews found that slight mechanical agita­tion of these eggs in sea water increased the number which developed. It has been shown in numerous experi­ments by Delage, R. S. Lillie, and the writer, that the substances causing development in the starfish egg are identical or closely related to those which bring about this effect in the egg of the sea urchin and in both cases the development is preceded by a membrane forma­tion.

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.[92]

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.

It is impossible to state at present whether the fertiliza­tion 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.[93] 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. Thus in 1901 Loeb[94] and Neilson showed that a short treatment with HCl and HNO3 sufficed to cause some eggs of Asterias in Woods Hole to develop into larvæ without a second treatment being needed, and Delage[95] showed the same for CO2; and in 1905 the writer found that the eggs of the Californian starfish Asterina can be induced to form a membrane by butyric acid treatment and that ten per cent. of these eggs developed into normal larvæ. Quite recently R. S. Lillie observed that the eggs of Asterias at Woods Hole can be caused to form membranes and develop into larvæ by a treatment with butyric acid and that the time of exposure required to get a maximal number of larvæ 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.[96]

All this leads us to the conclusion that the main effect of the spermato­zoön 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 spermato­zoön, 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 egg 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 171⁄2 to 221⁄2 minutes for Arbacia at 23° C.) in a solu­tion which is isosmotic with 50 c.c. sea water+8 c.c. 21⁄2 m NaCl,[97] they will develop into larvæ, 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 diminishes 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 1⁄10 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 concentra­tion required do not suppress or even lower the oxida­tions in the egg to any considerable extent,[98] 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 CO2 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.[99] 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.[100] 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., NaCl+CaCl2 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.

Herlant[101] 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 impossible.[102] 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 we 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.[103] In 1906 he found that the rapid disintegra­tion of the eggs of the sea urchin which follows artificial 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.[104] 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 spermato­zoön 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.[105] 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.[106] 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.

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.[107] He had found that when fertilized and unfertilized eggs were put into abnormal salt solu­tions, e. g., 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.[108]

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 CO2 but that the CO2 can escape from the egg as soon as its permeability is increased through the destruc­tion of the cortical layer of the egg.[109] After the CO2 has escaped, the excessive permeability must be restored to its normal value and this is the rôle 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 spermato­zoön enters. The cortical layer is removed along the cut surface and there is no reason why the CO2 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 CO2 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 half-hours 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. When 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 larvæ 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 larvæ. 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.

McClendon[110] has shown that the electrical conductivity of the egg is increased after fertiliza­tion, and J. Gray[111] 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 spermato­zoön 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.[112]

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 spermato­zoön 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. Delage[113] was courageous enough to make an attempt to raise partheno­genetic larvæ 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 spermato­zoön. 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.[114] (Figs. 7 and 8.) This would indicate that the frog belongs to those animals in which the male is hetero­zygous for sex.

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 spermato­zoön—although normally the egg of this form does not develop unless a spermato­zoön enters—corroborates the idea expressed in previous chapters that the egg is the future embryo and animal; and that the spermato­zoön, aside from its activating effect, only transmits Mendelian characters to the egg. The ques­tion arises: Is it possible to cause a spermato­zoön to develop into an embryo? The idea has been expressed that the egg was only the nutritive medium on which the spermato­zoön developed into an embryo, but this idea has been rendered untenable by the experi­ments on artificial parthenogenesis. Nevertheless the ques­tion whether or not the spermato­zoön 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 spermato­zoön 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. Incidentally 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 spermato­zoön underwent trans­forma­tion into a nucleus, but no mitosis or aster forma­tion was observed.[115] These experi­ments should be continued.