Artificial parthenogenesis and fertilization

Front matter

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Preface

In 1909 the author pubUshed a book entitled Die chemische Entwicklungserregung des tierischen Eies (Springer, Berhn), in which he gave an account of his experiments on artificial parthenogenesis. The object of these experiments was the substitution of phj'^sicochemical agencies for the mysterious complex ''living spermatozoon." The book has been translated into English by Mr. W. O. R. King, but, owing to the new observations since made, the author has found it necessarv to revise and enlarge the translation.

The book gives a survey of the methods by which the unfertilized egg can be caused to develop into an (^mbryo and the conclusions which can be drawn concerning the mechanism by which the spermatozoon produces this effect. The theory, which the author published in 1905 and 1906, that at least two factors are involved in this process, namely, one which brings about a change in the surface of the egg (the essential factor), and a second, corrective factor, seems to explain all the phenomena observed in the new territory and has proved a reliable guide.

In developing the new field of investigation, the writer endeavored to select those variables and methods which would lend themselves to a quantitative treatment.

The problem of fertilization is intimately connected with many different problems of physiology and pathology. We may mention, among others, the natural death of the egg cell and the prolongation of its life by fertilization; the fertilization of the egg by foreign blood and the innnunity of the egg to blood of its own species; the relations between heterogeneous hybridization and artificial parthenogenesis, between fertilization and cytolysis, and between permeability and physiological

vii v"i Preface efficiency of acids and bases. The facts recorded and discussed m the book therefore go beyond the special problem indicated by the title.

The Rockefeller Institute

FOR Medical Research

September 6, 1913

Contents

PAGE

I. Introductory Remarks ------ l

II. Some Remarks on the Morphology of Development - 17

II Some Remarks On The Morphology Of

DEVELOPMENT

Since this book is intended not only for the zoolojj;ist, hut more especially for the physiologist, pathologist, and chemist, it is necessary to give a sketch of the development of the animal egg. As an example we will use the egg of the sea-urchin, upon which the majority of experiments on the chemical activation

P C7

Fig. 1. — Unfertilized egg of the sea-urchin, S. pur pur at us, surrounded by spermatozoa.

Fig. 2. — The same egg about two minutes later, after the entrance of the spermatozoon and the formation of the fertilization membrane.

of development have been performed. The reason for this is to be found in the fact that the eggs of the sea-urchin can usually be obtained in large quantities and that they form the most suitable material for our problem.

Fig. 1 is a picture of the unfertilized egg surrounded by spermatozoa. (The flagella of the spermatozoa have been omitted in the drawing.) As soon as a spermatozoon has entered, a very characteristic alteration takes place in the egg; it becomes surrounded by the so-called fertilization meml)raiie (Fig. 2). The mechanism of this membrane formation can Ije more distinctly followed in the egg of Strongylocentrotus purpuratus by lowering the temperature of the sea-water; by this means, the process of membrane formation is retarded, and it

Fig. 3

Fig. 4

Figs. 3 and 4. — Origin of the fertilization membrane through the formation of small vesicles on the surface of the egg.

can be observed in its separate phases. Since we shall see later that this process of membrane formation is the essential part in the activation of the egg, we will trace it through its separate stages.

The beginning of the process shows itself in a roughening of the hitherto smooth surface of the egg (Fig. 3). This is due to the formation of countless tiny vesicles which stand

Fig. 5 Fig. 6 Fig. 7

Figs. 5, 6, and 7. — Further stages of the process of membrane formation.

out on the surface of the egg.^ These small droplets quickly increase in size (through absorption of sea-water) and flow together into larger drops (Fig. 4). This goes on (Figs. 5, 6, 7)

Hole.

1 1 have never observed this phenomenon in the eggs of Arbacia in Woods

Morphology of Development until finally the contents of all the drops have run together into a continuous layer around the egg^ (Fig. 2). Hence the surface lamellae of the tiny droplets form later the fertiHzation membrane.

At higher temperatures the process of memljrane formation in freshly removed eggs usually proceeds so quickly that the stages depicted in Figs. 3 to 7 are not distinctly seen and the ('ji;g passes directly from the condition of Fig. 1 to that of Fig. 2. At first the membrane adheres closely to the egg, and then of a sudden the space between the cytoplasm and the membrane increases enormously. In this latter way the process of membrane formation occurs in the egg of Arbacia.

After the formation of this fertilization membrane a second change takes place in the surface of the egg, inasmuch as a gelatinous film or membrane {G.M. Fig. 8) gradually appears on the surface of the cytoplasm. This gelatinous film does not form as suddenly or quickly as the fertilization membrane, but only begins to appear after ten minutes or more. The writer considers it possible that the formation of this film depends upon processes of oxidations, since its formation is delayed if the oxidations in the egg are retarded by the presence of KCN.

After membrane formation, the chemical processes which underlie development set in in the egg. The nuclear material grows and nuclear division occurs; this nuclear division, or rather the so-called nuclear spindle, is visible in the egg which we have chosen for description, viz., that of Strongylocentrotus purpuratus (Fig. 9). The spindle formation visible in this figure is immediately followed by cleavage or cell division, i.e., the partition

1 So-called "perivitelline space."

FiQ. 8. — Formation of a gelatinous flhn O.M. around the protoplasm of (lu> egg. about 15 minutes aftvr the formation of the fertilization membrane B.M.

of the egg into two spheres or cells. The various consecutive stages of this process are depicted in Figs. 10 to 13. First we see that the egg becomes somewhat elongated in the direction

of the axis of the spindle (Figs. 10, 11). This is probably due to the fact that the protoplasm flows to the poles of the spindle and away from its equator. Then there begins a cleavage of the protoplasm in the equatorial plane (Fig. 12), until the egg consists of two cells, each of which possesses a nucleus (Fig. 13). This process is called the segmentation of the egg.

We will now examine somewhat more closely this process of cell division or cleavage. We know two types of cell division ; one corresponds to the type of separation described here. The

Fig. 9. — Nuclear division (spindle formation) in the egg of S. purpuratus.

Fig. 10 Kig. 11

Figs. 10 and 11. — Beginning of segmentation in the egg.

second occurs in plants and consists in the formation of a solid membrane at the equator of the cell, without the two cells actually separating. At a certain stage, both types of divisions are identical, for in both certain materials are carried to the

Morphology of Development equatorial plane of the cell during the s])in(lle stage. In i)lant.s these materials consist of cellulose which hardens and forms a membrane separating the two cells. In animals they coasist of a different substance which possibly forms soaps (Quincke's albumin soap?) at the surface of the egg. These substances (soaps?) induce streaming phenomena, which according to the writer lead to the division of the egg into two spheres.^

Fig. 12 Fig. V.i

Figs. 12 and 13. — The first cell division completed.

T. B. Robertson has lately brought forward a pretty demonstration of this view.^ If a drop of olive oil is placed on the surface of water and upon one diameter of the drop there is laid a thread which has been previously moistened with an alkaline fluid (e.g. N/10 NaOH), the drop divides into two drops, just like the sea-urchin egg in the previously described figures. By suitably varying the viscosity of the oil and other conditions one can reproduce all the phases of cell division which can ])e observed at cleavage during the separation of the cells. The alkah on the thread forms a soap with the oleic acid, and this soap induces streaming phenomena which lead to the splitting of the drop into two.^ In discussing the possibility of a synthe-

iLoeb, Archiv f. Entwicklungsmechanik, I, 4:68-70, 189G; X.WII. i:V^. IWO; Butschli. ibid., X, 52. 1900.

-T. B. Robertson, Archiv f. Entwickhingsmechanik, XXVII. 29. 1909. 3 Loeb, The Dynamics of Living Matter, New York. 1906. pp. 5a-58.

sis of nucleins from lecithin, I have indicated that chohn must be set free in the hydrolysis of lecithin.^ Robertson assumes that this chohn, which is an alkah, may be the substance which serves to form the soap, and hence induces cell division.^ Obviously other alkalies may also be concerned. Perhaps in plants an acid substance — cellulose — is conveyed to the equatorial plane, and hence in this case a separation of the two cells does not take place, but only the formation of a solid separating membrane. This process of cell division is now repeated for each cell. From the two-cell stage (Fig. 13) the egg goes naturally to the four-cell stage (Fig. 14), eight-cell stage (Fig. 15), etc.

Fig. 14. — Four-cell stage of the egg of S. purpuratus.

Fig. 15. egg.

-Eight-cell stage of the

From the early stages there is a marked tendency for the single cells to creep to the surface of the egg; this may depend upon a tropism, perhaps a positive chemotropism of the cells toward oxygen. Owing to this creeping of the cells to the surface the first larval stage of the sea-urchin is a hollow sphere, the so-called blastula (Fig. 16). In this stage there appear on the outer surface of the cells cilia (which are omitted in Fig. 16)

1 Loeb, Ueber den chemischen Charakter des Befruchtungsvorgangs, und seine Bedeutung fur die Theorie der Lebenserscheinungen, Leipzig, 1908.

2 The interpretation of Robertson's experiment was combated by McClendon, Am. Jour. Physiol., XXVII, 240, 1910, and Archiv f. Entwicklungsmechanik,

XXXIV, 263, 1912. See also T. B. Robertson, Archiv f. Entwicklungsmechanik,

Morphology of Development by means of which the blastula swims about. At this period, the membrane is burst by some unknown influence, and the blastula, which at first swam around within the egg sheath

Fig 16. — Early blastula stage of the sea-urchin egg.

Fig. 17. — Gastrula stage of sea-urchin egg.

the

(fertilization membrane), now moves about freely in the water. The larva very soon rises to the surface. The next step in the development is the gastrula stage (Fig. 17). On one side the cells grow into the blastocele and this sac or tube growing into the cavity is the rudiment of the gut. On each side of the gut can be seen indicated the rudiment of the skeleton in the form of two crystals or triasters.

The further developmental stages consist in the organization of the gut into further divisions, and the outgrowth of the triasters into a larger skeleton. The originally spherical larva at the same time assumes a pyramidal form. It is called a pluteus (Fig. 18).

If the larvae are not fed, they live some 14 to 18 days at a temperature of about 15°. The further rearing is ver}^ tedious

Fig. 18. — The pluteus stage of the larva of S. purpuratus.

and difficult, as the right food of the larvae must be raised in the form of cultures. For our experiments we shall consider only the rearing of the pluteus stage. The kind of chemical activation of the egg, called in this book ''improved method," causes a development in the unfertilized sea-urchin egg which in a great number of cases corresponds to the form of development of the fertilized egg just described. The first division alone usually shows small irregularities, but these disappear in later divisions. On the whole, the reader can also apply the foregoing descriptions to artificial parthenogenesis.

The experiments described in this book were performed upon eggs such as are deposited by the female in the water. Usually males and females of these forms are found together in great numbers, and on certain days both sexes in one region simultaneously pour their sexual cells into the ocean. On the days on which a widely spread form spawns, the sea resembles a suspension of spermatozoa. The enormous numerical superiority of the spermatozoa over the eggs insures the fertilization of each egg. The view that the spermatozoon is chemotactically attracted by the egg apparently does not hold for the eggs of animals, although from time to time statements to the contrary have been published.

H. C. State College

Ill

1. Eighteen years ago the writer showed that if freshly fertihzed animal eggs (of sea-urchins and fishes) are deprived of all oxygen, no nuclear or cell division is possible.^ Godlewski^ and Samassa^ have found the same with frog eggs, and since then I have convinced myself that it holds for the eggs of the starfish, annelids, molluscs, and probably generally. In the same way oxygen is necessary for the maturation of the egg.

In these experiments on the prevention of development of the egg by lack of oxygen, one must guard against a source of error which lies in the fact that some time must elapse before the air or oxygen has been driven out of the dish containing the egg. Since in many forms the first division of the nucleus takes place in a little less than an hour, if the temperature is high enough, and since it often requires as long, or even a longer period, to replace all the oxygen by hydrogen, it may easily happen that a nuclear, or even a cell division, may take place after the egg has been placed in the stream of hydrogen.* I avoided this source of error by cooling with ice the vessel containing the eggs to 0° for as long as appeared necessary from the foregoing experiments to replace all the oxygen by hydrogen.

- Godlewski, " Die Einwirkung des Sauerstoflfs auf die Entwicklung von Rana, etc.," Archiv f. Entwicklungsmechanik, XI, 585, 1901.

3 Samassa, Verhandl. d. naturh.-med. Vereins zu Heidelberg, IV, 1898, and Verhandl. d. deutsch. Zool. Gesellsch., 1896 (quoted from Godlewski).

* It must not be taken for granted in general that oxygen is unimportant, if the experimenter does not succeed in suppressing all signs of life after a short passage of hydrogen or nitrogen through a vessel. It must not be forgotten that it takes a long time before the last trace of oxygen is driven out, and that often a trace of oxygen is quite sufficient to render possible the life phenomenon under investigation.

At this temperature the velocity of the chemical reactions in the egg is reduced almost to zero. On raising the temperature again, no division of the fertilized egg takes place in the hydrogen atmosphere; but on allowing air to enter the receptacle, the processes of nuclear and cell divisions are again at once resumed. Perhaps it can be still more strikingly demonstrated that oxidation is necessary for the development of the egg, by suppressing the oxidation processes by certain poisons. It has long been known that oxidations in the cell can be prevented by the addition of a little potassium cyanide, even when ox3^gen is present. I have found that the addition of 0.5 c.c. of a 1/20 per cent KCN solution to 50 c.c. of sea-water is sufficient to stop almost immediately the effect of the spermatozoon in the fertilized sea-urchin egg. When, however, such an egg has been transferred to normal sea-water and good aeration has been established, its development proceeds normally, provided that the eggs have not remained too long in the potassium cyanide solution.

From these facts the writer came to the conclusion that one essential effect of the entrance of the spermatozoon into the egg of the sea-urchin is the acceleration of processes of oxidation. ^ Oxidations go on also in the unfertilized egg. He concluded this from the fact that the unfertilized eggs in general disintegrate in a comparatively short time, while the addition of KCN or the withdrawal of oxygen prevented this disintegration, and he explained this phenomenon on the assumption that the oxidations in the unfertilized eggs accelerated their disintegration. The correctness of these ideas has since been proved by direct measurements.

O. Warburg was the first to measure the consumption of oxygen in the unfertilized and fertihzed sea-urchin egg, Arbacia

1 Loeb, "Ueber den chemischen Charakter des Befruchtungsvorgangs," Biochem. Zeitschr., I, 183, 1906, and also preface to Untersuchunuen tieber kunstliche Parthenogenese, Leipzig, 1906.

2 Loeb, Pfliiger's Archiv, XCIII, 59, 1902.

pustulosa, at Naples, by Winkler's method, and found that after fertiUzation the egg consumes from six to seven times as much oxygen as before fertihzation.^ Wastenej^s and I determined the consumption of oxygen in unfertihzed eggs of Arbada at Woods Hole. We found that immediately after fertilization the egg consumes almost four times as much oxygen as before fertilization.^ In experiments on Strongylocentrotus purpiiratus in Pacific Grove we found that immediately after fertilization the egg consumed five to seven times as much oxygen as before fertilization.^

This difference in the rate of oxidations in the unfertilized and fertilized eggs of the sea-urchin is probably due to the fact that the unfertilized egg of the sea-urchin is usually in the resting stage, i.e., no nuclear divisions are going on in it, when it is taken out of the ovary and it generally remains in this state if no spermatozoon enters.

2. The conditions are, however, entirely different in the starfish egg. This egg is, as a rule, immature when taken out of the ovary, but as soon as it gets into the sea-water, it may become mature; i.e., two nuclear divisions take place in succession and the polar bodies are thrown out. I have shown in a former paper that this process requires the presence of free oxygen in the same way as the developing sea-urchin egg. Lack of oxygen or presence of KCN prevents these nuclear maturation divisions in the starfish egg with the same certainty as it does the nuclear divisions in the fertilized sea-urchin egg.^ And, moreover, I was able to show that a slightly alkaline reaction of the surrounding solution is as favorable to the process of maturation of the starfish egg as it is to the segmentation of the fertilized egg of Strongylocentrotus purpiiratus.

1 O. Warburg, Zeitschr. f. physiol. Chem., LVII, 6. 1908. - Loeb and Wasteneys, Biochem. Zeitschr., XXXVI, 351, 1911. ' Loeb and Wasteneys, Jour. Biol. Chem., XIV, 469, 1913. « Loeb, "Maturation, Natural Death and the Prolongation of the Life of Unfertihzed Starfish Eggs," Biol. Bull., Ill, 295, 1902.

All these observations point to the conclusion that the processes determining or underlying nuclear division depend upon oxidations.

The eggs of the starfish must be fertilized at the time the second polar bod}^ is given off or immediately afterward, since otherwise they disintegrate and cannot be fertilized at all. When we compare the rate of oxidations in starfish eggs immediately before and after fertilization, we find no difference, as Table I shows. It should be remarked that while as a rule 100 per cent of the sea-urchin eggs can be fertilized by sperm, in the case of starfish eggs a much smaller percentage is usually fertilized since in most cases not all the eggs become mature simultaneously.

TABLE Ji

Number of Experiment

Consumption of

Oxygen of the Unfertilized Eggs

Consumption of

Oxygen of the

Fertihzed Eggs

Percentage of Fertilized Eggs

I

0.64

0.78

0.34

0.26

0.52

0.85

0.31

0.33

II

Ill

IV V

It is obvious that no noticeable increase in the rate of the oxidation is caused in this egg through the entrance of the spermatozoon. This is intelligible from the fact that those oxidations which lead to nuclear division were already going on in the eggs at the time the spermatozoon entered.

3. Warburg- compared the rates of oxidations in the eight-cell stage and the thirty-two-cell stage. Their ratio was as 4-2 to 6-8; a slight increase. Wasteneys and P measured the change in the rate of oxidations each hour for five consecutive hours after fertilization in the eggs of Arbacia at Woods

1 Loeb and Wasteneys, Archiv f. Entwicklungsmechanih, XXXV, 556, 1912.

2 Warburg, Zeitschr.f. physiol. Chem., LVII, 1, 1908.

1 Loeb and Wasteneys. Biochem. Zeitschr., XXXVI, .351, 1911.

Hole. The same eggs served as material for these determinations. Temperature, 23° C.

Consumption of oxygen per hour before fertilization 0.24 mg.

1st hour after fertilization 0.94 mg. 2d hour after fertilization 0.80 mg. 3d hour after fertihzation 0.87 mg. 4th hour after fertilization 0.91 mg. 5th hour after fertilization 1 .05 mg.

The value for the hour following fertilization is probably a little too high on account of the presence of sperm, which was washed away after the first determination. That this assumption is correct is shown by a repetition of the experiment.

Consumption of oxygen per U hours in fertilized egg —

first U hours 0.67 mg.

second 1| hours 0.74 mg.

third U hours 0.83 mg.

The consumption of oxygen increased about 24 per cent in 4 J hours. During this same time the eggs developed from the one-cell stage to the thirty-two-cell stage or beyond.

The question arises, What causes this increase ? The question cannot be answered definitely. During the division the total surface of the egg increases. But it must be remembered that the blastomeres are in close contact with each other and that hence only a fraction of their surface is exposed to the surrounding medium. If the intensity of oxidations increases with the total free surface of the eggs, the slight increase of the rate of oxidations with the process of segmentation might be

intelligible.

But this is not the only possibility. It had been stated by Boveri that in each cell division the mass of the nucleus doubled, so that in the eight-cell stage the total nuclear mass would be eight times as great as in the one-cell stage. But this has been denied by more recent workers like Miss Erdmann and Conklin.^

1 Conklin, "Cell Size and Nuclear Size," Jour. Exper. ZooL, XII, 1, 1912.

According to Conklin, the average nuclear growth during cleavage is not more than 5 per cent to 9 per cent for each division up to the thirty-two-cell stage. The possibility remains that the slight increase in the rate of oxidations wdth progressive cell divisions is due to the increase of the mass of the nuclei. This would be intelligible on the assumption that the rate of oxidation is in proportion to the mass of the nuclei. Such a view would harmonize with the suggestion expressed formerly by the writer, that the nucleus might be the main (although not the only) oxidizing organ of the cell.^

4. The observation that the nucleus of the fertilized egg remains unaltered during lack of oxygen or presence of potassum cyanide shows that oxidations are the prerequisites of the mechanical processes of nuclear and cell division. It can, however, be shown that aside from oxidations other chemical processes are accelerated in the sea-urchin egg by the process of fertilization. When fertilized eggs of Strong ylocentrotus purpuratus are left in sea- water free from oxygen for twenty-four hours at 15° C, they will not develop during that time, but they will begin to develop at once if oxygen is admitted. It \vill be found, however, that their development is no longer normal, since they form abnormal blastulae and never or rarely reach the gastrula stage. If unfertilized eggs are kept for twenty-four hours without oxygen they remain perfectly normal, since upon addition of sperm they develop normally and reach the pluteus stage. The result is the same if instead of withdrawing the oxygen we retard the oxidations by the addition of KCN.

Unfertilized and fertilized eggs of the same female were placed in a bowl with 50 c.c. of sea-water -f 2 c.c. 1/20 per cent KCN solution. 2 At different intervals, samples of these eggs

1 Loeb, Archie f. Entwicklungsmechanik, VIII, 689, 1899. Some authors attribute to me the opinion that in the protoplasm no oxidations occur. I have never expressed such an opinion.

2 Such a solution stops the segmentation in fertilized eggs.

were replaced in normal sea- water. Sperm was added to the unfertilized eggs after their transference to ordinary sea-water. The unfertilized eggs, which had been in the sea-water containing potassium cyanide for two days, developed in quite a normal manner, whereas the eggs which had been fertilized before they were put into the cyanide sea-water were no longer able to develop beyond the blastula stage after an exposure of only twenty-four hours to this solution. An exposure of five hours' duration to the cyanide sea-water was already harmful to the fertilized eggs; this was shown by the fact that while such eggs did develop after transference to ordinary sea-water, many larvae died during the first two days.^

These experiments leave no doubt that fertilization gives rise to a class of chemical reactions in the egg which can proceed independently of oxidations. It is very likely that other reactions besides oxidations, e.g., hydrolyses, take place in the egg. If such hydrolyses lead to the formation of harmful oxidizable substances, e.g., lactic acid, it can be understood why lack of oxygen must in time lead to the death of the fertilized egg; without oxygen the harmful substances, which are quickly rendered harmless by oxidation (or converted into substances like CO2, which can be^hminated), can now accumulate in the cell. Assuming that such hydrolyses are set up in the egg by fertilization, while they are lacking or are very slow in the unfertilized egg, we could understand why the fertilized egg suffers more quickly than the unfertilized egg from lack of ox3^gen or from the prevention of oxidations by potassium cyanide.

5. Wasteneys and the writer raised the question whether the oxidations are the independent variable in the development of the egg.^ From all we know, we should expect that hydrolj'ses

1 Loeb, "Versuche ueber den chemischen Charakter des Befruchtungsvorgangs," Biochem. Zeitschr., I, 189, 1906.

2 Loeb and W^asteneys, "Sind die Oxydationsvorgange die unabhiingige Variable in den Lebensersclieinungen ? " Biochem. Zeitschr., XXXVI, 345-56, 1911.

are the independent variable, and that the oxidations are determined or regulated by the hydrolytic processes. Our investigations do not contradict such a view. Since we are not able to measure the hydrolytic processes in the egg directly, we tried to solve our problem with the aid of the temperature coefficient. We determined the temperature coefficient for the velocity of segmentation in the egg of Arhacia by measuring the time which elapses from the moment of fertilization to the moment of the division of the egg into two cells for various temperatures. Then we measured the influence of the same variation of temperature upon the rate of oxidations in the cell. If the oxidations were the independent variable for the development of the egg the temperature coefficients for both processes should be identical or run parallel. This is, however, not the case. The experiments were made on the eggs of Arhacia at Woods Hole. The time which elapsed between fertilization and the first segmentation was as follows :

TABLE II

Temp. Deg. C.

m

18,

Time Required for

First Segmentation

4 +143*

3^+ 96^

2 + 831

2^+68

Temp. Deg. C.

Time Required for

First Segmentation

4 +43 3^+36^ 2 +31^

No segmentation; eggs suffered

* Where the times are given in the form of a sum the eggs were put into the thermostat as many minutes after fertilization as the first figiu"e indicates.

The temperature coefficients for segmentation are the greater the lower the temperature. For the interval 7°-17° the coefficient is more than three times as large as for the interval 17.5°-27.5° (Table III).

TABLE III

Interval of Temperature

Temperature Coefficient for 10°C. for the Velocity of Segmentation in the Egg of Arbacia

Interval of Temperature

Temperature Coefficient for 10°C. for the Velocity of Segmentation in the Egg ot Arbacia

7°-1 7°

7.3

6.0

>4.0

~3.9

3.3

15°-25°

2.6

16°-26°

2.6

9°-19°

17.5°-27.5°

20°-30°

2.2

1 0°-20°

1.7

12°-22°

A determination of the temperature coefficient of oxidations in the egg of Arbacia did not show such a variation.

TABLE IV

Interval of Temperature

Temperature Coefficient for 10° for the Velocity of Oxidations

Interval of Temperature

Temperature Coefficient for 10° for the Velocity of Oxidations

15°-25°

2.24

'^° 1 ^°

17°-27°

2.00

') 1.0 • •

7° 17°

20°-30°

1.96

10° '>0°

22°-32°

1.40

13°-23"

The temperature coefficients of oxidations for a difference of 10° C. are practically identical throughout. For the interval 7°-17° the coefficient is 2.0 and for 17°-27° it is also 2.0. This result points toward the probability that the rate of cell division is not directly determined by the rate of oxidations, although the possibility exists that besides the influence of temperature upon the velocity of chemical reactions an influence upon some physical property of the egg is superposed (e.g., viscosity) which makes itself felt only or chiefly at the lower temperatures and becomes the stronger the lower the temperature.

6. The writer has shown that segmentation and development can go on only if the concentration of the HO ions in the surrounding solution reaches a certain height; this height is greater for the egg of Strongylocentrotus purpuratus than for Arbacia.

vSea-water is, according to van't Hoff, a mixture of the following composition : 100 molecules NaCl, 2 . 2 molecules KCl, 1.5 molecules CaClg, 7.8 molecules MgClg, and 3.8 molecules MgS04.' To this, traces of NaHCOs and Na2HP04 are to be added. The osmotic pressure of the ocean water show^s local variations. The eggs of the animals in Pacific Grove develop best if the salts are used in a half grammolecular concentration. For the fauna in Woods Hole, on the Atlantic, a slightly higher concentration, about 21/40 m is, perhaps, the optimum, although m/2 solutions give almost the same result. Solutions of 25/40 m are decidedly injurious.