The Dynamics of Living Matter

O. and R. Hertwig discovered that if sea water is saturated with chloroform -- only traces of which are soluble in water --the unfertil- ized eggs of the sea urchin form a membrane when put into this chloro- form sea water.* Herbst found that benzol, toluol and creosote act similarly. f It seemed to me that possibly hydrocarbons in general might act in this way, and as a test I used amylene. It indeed called forth the membrane formation. This method of calling forth a mem- brane formation by hydrocarbons has a serious drawback, inasmuch as the eggs show a tendency to undergo cytolysis, and are killed. By the speedy transportation of the eggs into normal sea water some may be saved.

It seemed of interest to ascertain whether it made any difference for the parthenogenetic development which substance was used for the pro- duction of the membrane. When the eggs were taken immediately after the formation of the membrane from the sea water containing benzol, not all the eggs that had formed a membrane underwent cytolysis. When these eggs were subsequently treated in the way described above with hypertonic sea water, they segmented, and some of them developed into plutei. As long as the formation of a membrane is induced by a substance which does not injure the egg too much, the subsequent short exposure to hypertonic sea water may lead to the formation of an em- bryo. In regard to their vitality, and possibly their structure, the em- bryos may possibly differ according to the substance which is used for the production of the membrane. This, however, must be determined by further experiments.

It agrees further with the idea that the membrane formation and not * O. and R. Hertwig, Untersuchungen zur Morphologic und Physio logie der Zelle, Heft 5, Jena, 1887. the acid effect is essential in these experiments, that not all the acids can be used in these experiments. HNO3, HC1, H2SO4 and dibasic or tribasic organic acids such as oxalic or citric, etc., could not be used, while CO2 called forth the membrane formation. The order in which the two agencies are employed is not a matter of indifference. When the eggs are first exposed to the above-mentioned hypertonic sea water for about twenty to fifty minutes and then sub- mitted to a process which calls forth the membrane formation (e.g. to the treatment with fatty acid), the eggs form a membrane, but will not develop into larvae. As a rule, they disintegrate within twenty-four hours, and behave in every way as if they had been treated with the acid alone. If one wishes to treat them with hypertonic sea water first, they must remain in this solution for about from one and one half to two hours. If after this time the membrane formation is called forth, almost all the eggs develop, and a number of the larvae rise to the sur- face. This method also gives good results.

If in eggs the membrane formation is called forth first, and if they are subsequently exposed to the above-mentioned hypertonic sea water for more than fifty minutes, either no egg develops or the development is very abnormal. All the facts mentioned in this and the previous communications indicate that the process of membrane formation is an essential and not a secondary phenomenon in this method of artificial partheno- genesis. Five years ago I ventured the suggestion that the process of membrane formation is a process of coagulation. It is, however, obvious that the membrane formation in these experiments cannot be attributed to an acid coagulation, as in this case the membrane formation should occur while the eggs are in the acid, and not after they are taken out. Moreover, the fact that only certain acids act in this way also excludes such an opinion. These facts suggested the possibility that the fatty acids did not produce the membrane forma- tion through the H-ion, but by the anion or the undissociated molecule, and that, moreover, the H-ion directly antagonized the membrane formation. This idea was tested and found correct. If a fatty acid is added to benzol sea water, the eggs are no longer able to form a mem- brane while they are in this mixture, though they form a membrane while they are in benzol sea water which is free from fatty acid. From a closer observation of the process of membrane formation I am in- clined to believe that it is due to a process of secretion, i.e. the squeez- ing out under pressure of a liquid from the interior of the egg.* I am

no more able to state the nature of the forces which underlie secretion in this than in any other case. These experiments show that it is possible to completely imitate by physicochemical means the effect of the spermatozoon upon the sea, urchin egg. It is also obvious that this method is somewhat complicated and specific, and that it cannot be well covered by the phrase that the method consists in a "stimulation," for the word "stimulation " does not — as far as I know — mean that we have to treat an organ first for one

half minute with 50 c.c. of sea water + 3 c.c. - - butyric acid and then for from twenty-five to fifty minutes with a mixture of 100 c.c. of sea water +14 c.c. 2\ n NaCl solution. Moreover, these quantitative data vary slightly for different species of sea urchins, e.g. Strongylocentrotus purpuratus and jranciscanus. Yet some authors have maintained that any kind of stimulus, or various chemical substances, might produce artificial parthenogenesis in the egg of the sea urchin. These statements are based partly on mis- understandings and partly on errors. In a former paper I stated that it makes no difference how the osmotic pressure of the sea water is raised, whether by sugar, by urea, or by salts; if the pressure is only sufficiently high, the parthenogenetic development of the sea urchin's egg will occur. Morgan makes use of this fact to attempt to show that inasmuch as sugar as well as salts cause the development, various stimuli can produce the development. He overlooks the fact that in this case the sugar or salt does not act chemically, but solely osmotically by with- drawing water from the egg, and that for this effect it is immaterial what the chemical character of the dissolved substance is. Other authors have been misled by mistaking parasitic larvae found in their cultures for the larvog of sea urchins. Ariola has maintained that the eggs of sea urchins develop normally parthenogenetically at Naples. I mav state that neither the unfertilized eggs of Arbacia nor those of

Strongylocentrotus of the Atlantic or the Pacific coast of America ever develop, and that the same has been found for the eggs of the sea urchins at Naples by all competent workers. Ariola has given a description and drawings of the larvae he found which he considered as normally parthenogenetic larvae of sea urchins, and I believe that they were prob- ably larvae of some mollusk; they were certainly not the larvas of the sea urchin. I mention this fact simply to show that unless an author actually observes the origin of a larva from the egg, he may fall into serious error. Viguier maintains that the sea urchins in Algiers are naturally parthenogenetic. I should place more confidence in this author's statements were they written in a more dispassionate, scien-

tific tone, and if his precautions against sources of error were more adequate. Still, it is not impossible that some physical or chemical condition accidentally present in Algiers may bring about effects similar to the extraction of water from the eggs of these animals in this country. Since it is possible to fertilize the egg of the sea urchin and that of the starfish by the spermatozoa of the latter species, it seemed also pos- sible that the fertilization of the starfish's egg might be caused by the same substances which cause the fertilization of the egg of the sea urchin. I have made experiments on the egg of a form of Asterina which is com- mon in the bay of Monterey.* This egg forms a membrane upon the en- trance of a spermatozoon. I found that as in the case of the sea-urchin egg, the egg of Asterina forms a membrane after having been treated with a fatty acid. The only difference is that the egg of Asterina requires more acid for this result than the egg of Strongylocentrotus. When the eggs of Asterina had been put for about one and one half to two minutes into a

n mixture of 50 c.c. sea water + 5 c.c. - - acetic or butyric acid, they formed a membrane when put back into normal sea water. When they were put into 50 c.c. sea water + i c.c. benzol or amylene, they formed a membrane while they were in this mixture. Eggs in which this membrane formation had been called forth were able to develop into normal larvas, and the development of such eggs resembled in rapidity and the form of the larva? completely that pro- duced by sperm.

The egg of the starfish is, as a rule, not mature when it leaves the ovary. It possesses a large nucleus, and the process of maturation con- sists in the nucleus being dissolved in the protoplasm of the egg and the polar bodies being thrown out. As long as the large nucleus is visible in the egg it cannot be fertilized by a spermatozoon, nor can its develop- ment be called forth by a treatment with one of the fatty acids or with one of the hydrocarbons, like benzol or amylene. Not until the nucleus has become dissolved in the protoplasm can a spermatozoon fertilize the egg, and at about the same time it becomes possible to produce artifi- cially a membrane formation and development.

There is a noticeable difference in the method by which the starfish egg can be caused to develop and the method which is necessary in the case of the sea-urchin egg. For the former the process of artificial mem- brane formation is sufficient, while the sea-urchin egg has, in addition, to be submitted for a short time to the action of hypertonic sea water. This difference is rendered a little more comprehensible by the fact that a small percentage of the eggs of the starfish are able to develop without any external cause or agency being applied. The number of these eggs varies in the eggs of different individuals, but is, as a rule, very small, e.g. a fraction of i per cent. The rate of segmentation in these "natu- ; rally" parthenogenetic eggs is slower than the rate of development of the eggs fertilized by sperm, and the blastula begins to swim consid- erably later than the blastulae coming from fertilized eggs or from eggs in which a membrane had been produced artificially. Moreover, the blastulae of the spontaneously developing eggs differ somewhat in ap- pearance from the blastulaa coming from the two latter.

Neilson and I found that the number of eggs which develop without a membrane formation can be increased by treating the eggs transitorily with acidulated sea water. Delage* simultaneously obtained the same result by treating the eggs of Asterias with CO2 ; I am inclined to be- lieve that the CO2 acts as an acid, although Delage is not willing to admit this. The fact that the unfertilized eggs of the starfish may develop with- out any external cause has often been overlooked, and this has led some authors again to state that any "stimulus" may cause the development of this egg. Acids, indeed, increase the number of eggs which will develop; the same is possibly true for mechanical agitation, as A. P. Mathews has observed. f He is inclined to believe that in this case the mechanical agitation is the direct cause of development (by producing coagulation). It is, however, necessary to state that the taking up and dropping of eggs with a pipette suffices. In eggs of Amphitrite, an Annelid, I have convinced myself that the number of eggs which develop does not bear any relation to the extent of the mechanical agitation. I consider it possible that some secondary factor connected with the agi- tation, such as the diffusion of gases into or from the egg, e.g. CO,,, may be the real factor involved in this case.

The experiments thus far mentioned indicate that the process of membrane formation, or some process underlying this, is of importance for the complete physicochemical imitation of the developmental in- fluence of the spermatozoon. The question arises, What is the nature of this process? It seems to me from my observations on Echino- derms that the essential feature of this process is the squeezing out under pressure or the secretion of a fluid from the protoplasm of the egg. As a mechanical effect, the surface film of the egg is lifted and separated from the protoplasm by a liquid secreted by the egg. When the secretion of this liquid occurs very slowly, the lifting up of the sur-

face film will not occur; only when the secretion of the liquid is rapid enough will the secretion result in the membrane formation. According to this view, the secretion of a liquid from the egg is the essential feature, while the membrane formation itself is possibly only a secondary, mechan- ical effect of the sudden secretion. If this be true, the essential feature in fertilization in Echinoderms is not the membrane formation itself, but the secretion of a liquid from the interior of the egg. This conception is corroborated by an observation I made several years ago. I found that the unfertilized eggs of a sea urchin could be kept alive in sterilized sea water for a week, or possibly more. When sperm was added to such eggs, they developed, but without the formation of membranes. It is quite possible that a process of secretion may be produced in every egg through the entrance of a spermatozoon, while the actual separation of the surface film of the egg from the protoplasm is only a secondary mechanical consequence of this secretion, which may or may not occur. Since I have only recently recognized the importance of the process of membrane formation for the complete physicochemical imitation of the developmental effect of the spermatozoon, I have not yet found time to see whether it holds good only for Echinoderms. An observation re- cently made by Professor Lefevre on artificial parthenogenesis in a marine worm, seems to indicate that the artificial membrane formation, or rather the process underlying it, is of more general importance. Lefevre found that about 50 per cent of the eggs of Thalassema develop into normal larvae, after having been exposed to sea water (to which a little acid had been added) for a few minutes. After they were taken out of the acidulated sea water, they formed a membrane and developed. The case seems to be similar to that of A sterina.

The development of the eggs seemed to be normal, and the vitality of the larvae seemed to be the same as that of the larvae originating from fertilized eggs. I, as well as others, had, before Lefevre's observations, produced artificial parthenogenesis in the eggs of worms, but without artificial mem- brane formation. In all these cases the larvae had always a diminished vitality, and the development was often different from that of the egg fertilized by sperm. I had found that the unfertilized eggs of Chtztopte- rus, a marine Annelid, can be caused to develop into swimming larvae* with certainty by adding a small but definite amount of a soluble potas- sium salt ; but the vitality of these eggs was considerably less than that of the larvae originating from fertilized eggs. I may also add -- although this does not belong to our problem - - that I noticed that the eggs of Chcetopterus, which had been caused to develop parthenogenetically by KC1, reached the trochophore stage and began to swim about seem-

ingly without segmenting. Frank Lillie* afterward examined such eggs histologically and convinced himself indeed that such is the case. Bullotf produced a much more normal type of development in the un- fertilized eggs of another Annelid, Ophelia, by submitting these eggs for about two hours to hypertonic sea water. In this case the segmen- tation was normal and the larvae formed were also normal, but they only lived two days. It will therefore be of interest to find out whether in ChcBtopterus the same means which in Echinoderms lead to a mem- brane formation are able to induce a parthenogenetic development which resembles in all its features the development caused by a spermatozoon.

Kostanecki | found that by a treatment with hypertonic sea water, the unfertilized eggs of a Mollusk (Mactra} could be caused to undergo the first segmentations; and I found afterward § that this method led in other Mollusks (Lotlia, Acnma) to the production of swimming larvae. I have recently tried my new method on the eggs of Lottia gigantea. It seems that the combination of the treatment with fatty acid and hypertonic sea water gives better results than the osmotic treatment alone. Some attempts have been made to cause the eggs of vertebrates to develop parthenogenetically. Bataillon|| has shown that the unfer- tilized eggs of the frog and of Petromyzon can be caused to segment as far as the morula stage by putting them for some time into a salt solu- tion of a certain concentration, whereby they lose water.

It was natural to try whether or not substances can be extracted from the spermatozoon which cause the unfertilized egg to develop. Fieri made the statement that this could be done, but he evidently worked with sea water contaminated by spermatozoa. After the appearance of my first paper, H. Winkler made experiments with the extract of spermatozoa of sea urchins which, according to his description, caused the eggs of the same species to go through the first stages of segmenta- tion; If no larvae, however, developed from these eggs. These experi- ments were repeated by Gies, who tried to ascertain whether or not an enzyme could be obtained from the spermatozoon which caused the unfertilized egg to develop; but the results were absolutely negative. Not a trace of segmentation could be produced in eggs treated with such extracts. These results contradict the conclusions of Winkler.** I am inclined to believe that Winkler worked with sea water whose concen-

§ Loeb, University of California Publications, Physiology, Vol. I, p. 7, 1903. If Hans Winkler, Nachrichten der Gesellsch. der IVissenschaften zu Gottingen, p. 87, 1900. tration had been slightly raised, or which had been rendered slightly alkaline through evaporation. In either case results such as he produced may be observed. Max Cremer also obtained absolutely negative results when he tried to cause the development of fish eggs with extracts obtained from the sperm of the same species with the Buchner press.

We may finally raise the question whether we can form, on the basis of the facts mentioned, any idea as to how the spermatozoon causes the egg to develop. From the facts stated in our fourth lecture in regard to cell division it is obvious that the essential effect of the spermatozoon consists in the transformation of part of the protoplasmic or reserve material in the egg into the specific nuclein or chromatin substance of the nucleus. In each nuclear division one half of the mass of each original chromosome goes into the nucleus of each of the two resulting cells. But during the resting period which elapses until these nuclei are ready to divide again, each chromosome grows to its original size again, and then a new division occurs. It is quite possible that the oxygen which is required for the process of cell division is needed for the synthesis of nuclein or chromatin substance. The fact that the rate of development is influenced by temperature in much the same way as are chemical reactions supports the idea given above that the essential feature of fertilization consists in the starting or the acceleration of a chemical reaction which is going on steadily in the egg.

It was natural to think first of the possibility that the spermatozoon carries a positive catalyzer into the egg, and thus accelerates the above- mentioned synthetical process, which might also occur in the unferti- lized egg but too slowly to lead to any development. It occurred to me that if this idea were correct the unfertilized eggs of the sea urchin'might segment in normal sea water if they only could be kept alive for a suffi- cient length of time. In order to test this idea I took out the ovaries with bacteriological precautions and kept the eggs alive in sterile sea water for a week. Not an egg segmented, but when sperm was added, seg- mentation occurred promptly. This observation did certainly not support my idea of the spermatozoon carrying a positive catalyzer into the egg. It then occurred to me that a rise in temperature should act like a spermatozoon, since a rise in temperature should accelerate the velocity of chemical reactions. While a rise in temperature promptly accelerates the development after the egg is fertilized, or caused to de- velop by physicochemical methods, I have thus far not been able to start development in this way.

It then occurred to me that a superposition of two methods of fer- tilization should lead to an acceleration of a process of development, if it were true that the nature of fertilization consisted in a positive cataly- sis. I combined fertilization by sperm, osmotic fertilization, and the new method of fertilization in all possible ways in the egg of Stron- gylocentrotus without, however, being able to accelerate the process of development ; on the contrary, as a rule, the process of development was markedly retarded. The idea that the spermatozoon carries a positive catalyzer into the egg has, therefore, thus far not received any support.

A second possibility which was to be considered was that the sper- matozoon removes from the egg somehow a negative catalyzer or a condi- tion whose presence in the egg prevents the development of the latter. If this were the case, we could readily understand why a rise in temperature which accelerates the development in the fertilized egg cannot - - as far as my present knowledge goes — start the process of development in the unfertilized egg. We can, moreover, well understand why a process of secretion which seems to underlie the membrane formation may be of such great importance for the process of development. Finally, we may be able to understand a fact which I have observed in the eggs of starfish, and which has not yet been mentioned. When the eggs of Asterina or Aslerias are allowed to ripen, they will die within a few hours unless they develop either spontaneously or through the influence of sperm or some of the above-mentioned agencies.* The disintegration which leads to the death of the nondeveloping egg is obviously due to an oxidation, since I found that the same eggs when kept in the absence of oxygen will not disintegrate. We know that oxygen is an absolute prerequisite for the development of the fertilized egg. The fact that oxygen is a poison for the mature but nondeveloping egg shows that altogether different chemical processes must occur in the unfertilized, nondeveloping and the developing egg of the starfish. The process of fertilization seems, therefore, to consist in the elimination or alteration of a chemical condition in the egg, and that this alteration makes the processes of synthesis of nuclein material from the protoplasm possible.

In my first experiments on artificial parthenogenesis I was inclined to believe that the immediate effect of the methods employed consisted in a modification of the condition of the colloids in the egg. This view is contradicted by my recent experiments. When the process of arti- ficial membrane formation is produced in the egg of a sea urchin, the egg does not show the changes leading to a cell division, e.g. the forma- tion of astrospheres, until after one or two hours. But even this does not

lead to the development of the egg unless the egg has been submitted for twenty minutes to the hypertonic sea water. It is therefore obvious that the process of astrosphere formation or similar alterations cannot be the direct effect of the act of fertilization and, moreover, it cannot be the essential feature of it. I am inclined to believe that the direct and essential effect of the spermatozoon and the methods of artificial par- thenogenesis is the starting of a definite chemical process, and that the formation of astrospheres is only a secondary effect of this.

It is in harmony with this idea that the process of segmentation in the case of artificial parthenogenesis is entirely regular, and does not differ from that of fertilized eggs, provided that the right concentration and time of exposure are selected. I have not entered into a discussion of the cytological changes which are noticeable in an egg in which artificial parthenogenesis has been produced, and refer the reader to a masterly paper* by E. B. Wilson on this subject.

IN addition to the developmental effects, the spermatozoon has a hereditary effect, inasmuch as it transmits the paternal qualities to the offspring. The experiments on artificial parthenogenesis or chemical fertilization suggest the possibility that the developmental and the heredi- tary agencies in the spermatozoon are connected with different substances. O. Hertwig twenty years ago denned the process of fertilization as the fusion of two nuclei; namely, the egg nucleus and the sperm nucleus. While this fusion is apparently of importance for the hereditary effects, one fails to see how a fusion of two nuclei must cause an egg to develop. The experiments on artificial parthenogenesis indicate clearly enough that the development of the egg can be caused without even the presence of a sperm nucleus. On the other hand, the experiments on merogony show that a fragment of egg protoplasm which has no nucleus can develop when fertilized by a spermatozoon. Delage made extensive experiments in which he cut pieces of protoplasm from the egg of Echinoderms, Annelids, and Mollusks.* These pieces developed when a spermatozoon entered into them. In this case fertilization occurred without a fusion of nuclei, as there was no egg nucleus present.

It is a very striking fact that for the first stages of development the hereditary influences of the spermatozoon and the egg are by no means equal. It seems that for these first stages the influence of the egg by far exceeds that of the spermatozoon. It may almost be said that the first stages of the embryo are exclusively or almost exclusively determined by the egg, and not by the spermatozoon. This is best illustrated if we hybridize forms whose first stages of development differ radically from each other, e.g. sea urchin and starfish. The pure larvae of both forms go through a blastula and gastrula stage, but then their development becomes strikingly different, inasmuch as the sea urchin larva develops into a pluteus with a skeleton, while the starfish larva

forms no skeleton. If the egg of a sea urchin is fertilized by the sperm of a starfish, those larvae that live long enough develop invariably into a pluteus.* It would be interesting to ascertain whether the hybrid larvae produced from a starfish egg by the fertilization with a sea urchin spermatozoon ever form a pluteus larva. These data also indicate that the statement that fertilization consists in the fusion of two nuclei does not cover all the facts.

As far as the adult is concerned, it seems that, as a rule, spermato- zoon and egg have an equal share in the transmission of the hereditary qualities. Mendel states that in the case of the hybridization of two species of peas, a and b, the results were the same, whether the pollen came from the species a and the egg from the species b, or vice versa. We shall see later on that the early embryo is to a certain extent predeter- mined in the protoplasm of the egg. This makes it natural that these early stages should depend upon the egg, and not upon the spermato- zoon. As far as the adult is concerned, the protoplasm has to be formed by the taking up of food, and the chemical as well as the subsequent physical changes which the material undergoes will be under the in- fluence of the catalytic agencies of both the egg and the spermatozoon.