the influence of hypertonic solution upon · the influence of hypertonic solution upon the rate of...

THE INFLUENCE OF HYPERTONIC SOLUTION UPON THE RATE OF OXIDATIONS IN FERTILIZED

AND UNFERTILIZED EGGS.

BY JACQUES LOEB AND HARDOLPH WASTENEYS.

(From the Rockefeller Institute for Medical Research, New York.)

(Received for publication, April 21, 1913.)

I.

In a series of papers published since 1905 one of the writers has shown that in the egg of the sea urchin the initiation of normal development requires two different agencies. The one is needed to call forth a typical change in the surface of the egg which results in the formation of a more or less typical “fertilization membrane. ” This change causes the egg to segment and if the temperature is low some of these eggs may reach an early larval stage. At room temperature, however, the eggs begin to disintegrate after artificial membrane formation, as a rule, during the first cell division. These facts prove that the artificial membrane formation (e.g., by butyric acid) suffices to set the whole machinery of cell division and development into motion but that the egg is sickly and disintegrates the more rapidly the higher the temperature.l

It was shown by the same author that this disintegration after artificial membrane formation is retarded or suppressed if we deprive the egg of oxygen or if we retard the rate of oxidations in the egg through the addition of a trace of KCN. The addition of a sufficient amount of chloral hydrate (or probably any other narcotic) acts in the same way, although chloral hydrate does not lower the rate of oxidations. But all these agencies have one object in common, namely, that they inhibit the processes of nuclear and cell division. The disintegration of the unfertilized

1 Loeb: Die chemische Entwicklungserregung des tierischen Eies, Berlin. 1909; The Mechanistic Conception oj’ Life, 1912.

469

by guest on February 15, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

470 Effect of Hypertonic Solution on Oxidations

egg after membrane formation is therefore connected either with oxidations or with the nuclear and cell divisions consequent upon oxidations. We can cure the egg from this disease by one of two agencies : We either put it for a short time (from 30 to 60 minutes) into a neutral hypertonic solution; or we put it for a longer t’ime (about 3 hours) into sea water which is free from oxygen or which contains some KCN. The former method gives more uniform results. Such eggs develop into larvae at room temperature.

These results gained in importance since it could be shown that the developmental effect of the spermatozoon is also due to two different agencies, one of which causes merely membrane formation while the other produces the corrective effect.2

Warburg had already found that the artificial membrane formation raises the rate of oxidations in the egg to the same height as the entrance of the spermatozoon, as was to be expected and as we were able to confirm.3 Since Loeb had found that the hypertonic solution acts only in the presence of free oxygen and t’hat its action is suppressed by the addition of a t’race of KCN, it was of interest to find out whether or not the hypertonic solution alters the rate of oxidations in the egg after artificial membrane formation. The experiments were made in this way, that the rate of oxidations in the eggs, after artificial membrane formation, was determined, first in normal sea water and later in hypertonic solution.

These experiments are by no means simple, since it is necessary that all the eggs possess membranes; for we shall see aft’erwards that, in unfertilized eggs without membranes the hypertonic solution causes a decided rise in the rate of oxidations.

The unfertilized eggs of one female, Strongylocentrotus purpuratus, were divided into six equal parts. Two remained unaltered and served as checks. The eggs of two lots were treated with butyric acid and all formed membranes. One of these lots was put into normal sea water, the other into hypertonic sea water (50 cc. sea water + 8 cc. ?,F NaCl) such as is used to cause the eggs to develop normally after artificial membrane formation. The fifth lot was fertilized wit,h sperm. The sixth lot served for some other experiment which does not concern us here. The eggs remained in each solution one hour. Temperature 18°C.

2 Loeb: lot. cit. 3 Warburg: Zeitsclr. f. physiol. Chem., lxvi, p. 305, 1910.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

J. Loeb and H. Wasteneys

TABLE I.

1. Unfertilized eggs in normal sea water (con- trol 1). . . . . .

2. Unfertilized eggs in normal sea water (con- trol2).....................................

3. Unfertilized eggs after membrane formation in normal sea water.

4. Unfertilized eggs after membrane formation in hypertonic sea water.,

5. Fertilized eggs in normal sea water.. .

mgm.

0.12

0.12

0.53

0.54 0.57

471

COEFFlCIENT ,*1 OXIDATIONB

__..~

1.00

1.00

4.40

4.50 4.70

It is obvious that the hypertonic solution does not increase the rate of oxidations in the unfertilized eggs after artificial membrane formation. Table II gives the result of a second experiment of the same kind. Temperature 15°C.

TABLE II.

1. Unfertilized eggs in normal sea water.. . . . 2. Unfertilized eggs after membrane format.ion

in normalseawater........................ 3. Unfertilized eggs after membrane formation

in hypertonic sea water. . . 4. Fertilized eggs in normal sea water.. . .

mgm.

0.18

0.85 4.72

0.88 4.88 0.82 4.55

1.00

The result is identical with that in table I. Membrane formation raises the rate of oxidations t,o the same height as fertilization, but the subsequent treatment of these eggs with the hypertonic solution has no effect upon the rate of oxidations. This experiment was repeated three times with t’he same results as shown in Table III. The oxygen consumption is always for one hour at 18°C.

All these experiments prove conclusively that the curative effect of the hypertonic solution after the artificial membrane formation is not due to an increase in the rate of oxidations in the egg. It cannot be said, however, that it is independent of oxidations,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


TABLE III.

Unfertilized eggs after membrane formation in normal sea water. :

Unfertilized eggs after membrane formation in hypertonic sea water.

Unfertilized eggs after membrane formation in normal sea water. .


Unfertilized eggs after membrane formation in normal sea water.


mgm.

0.83

0.74

0.52

0.54

0.74

0.70

1.00

0.90

1.00

1.04

1.00

0.90

since the curative effect of the hypertonic solution is retarded or suppressed if the oxidations in the egg are suppressed. Loeb formerly suggested that through the exposure to the hypertonic solution an oxidation product is formed in the egg whereby the latter is saved from the threatening disintegration. It may be that an injurious substance contained in the egg after membrane formation is destroyed or that a new substance lacking in the egg is supplied. The same curative effect can be produced more slowly through other processes in the egg which take place in the absence of oxygen.

II. THE INFLUENCE OF HYPERTONIC SOLUTIONS UPON THE RATE

OF OXIDATIONS IN FERTILIZED EGGS.

Since the unfertilized egg after artificial membrane formation behaves in regard to oxidations like a fertilized egg, it was of interest to find out whether or not the hypertonic solution accelerates the rate of oxidat’ions in eggs fertilized by sperm. 0. Warburg states that this is the case, and that the increase may be 300 per cent.4 We absolutely failed to notice any increase in the rate of oxidations when the eggs of S. purpuratus were put into hypertonic sea water, no matter how great the degree of hypertonicity. In

( Warburg: Zeitschr. j. physiol Chem. lx, p. 442, 1909.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

J. Loeb and H. Wasteneys 473

part of the experiments, 1-3, the rate of oxidations was successively measured in the same eggs for one and a half hours in normal sea water, and then for one hour in the hypertonic sea water. In the experiments 4-7 the eggs of the same female were divided into eight equal parts; four of these were put into normal sea water, the others into hypertonic sea water of different concentrations. This experiment may incidentally also serve as a check for the accuracy of the method.

The temperature was 18°C. and the consumption of oxygen measured for one and a half hours. Table IV gives the results.

There can be no doubt about the fact that the hypertonic solution does not increase the rate of oxidations in the fertilized egg of ,‘trongylocentrotus pwpuratus. This harmonizes with our previous

1

2

3

4

5

6

7

TrlBLE 11..

Fertilized eggs in normal sea water. Fertilized eggs in 50 cc. normal sea

water + 8 cc. q NaCl..

Fertilized eggs, in normal sea water. Fertilized eggs in 50 cc. normal sea

water+8cc.+NaCl...........


water+ 8 cc.FNaCl...........


water + 4cc.%NaCl...........

Fertilized eggs in.normal sea water. Fertilized eggs in 50 cc. normal sea

water + 12cc.~NaCl........__


water + 12 cc. -$NaCl.


water + 16 cc. ?FNaCl.. .

mgm.

0.87 1.00

0.86 0.99

0.60 1.00

0.52 0.87

0.55 1.00

0.59

1.30

1.27

1.30

1.54

1.33

1.53

1.33

1.57

1.07

1.00

0.98

1.00

1.20

1.00

1.20

1.00

1.20


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


result that the hypertonic solution does not increase the rate of oxidations in unfertilized eggs of the same species after artificial membrane formation, since the membrane formation is t.he essential feature in the causation of development by sperm or by artificial means.

III.

The fact that. a hypertonic solution does not increase the rate of oxidations in the unfertilized eggs after artificial membrane formation seems at first sight to contradict an observation made by Warburg at Naples, that the hypertonic solution raised the rate of oxidations in unfertilized eggs (which have not been submitted to the process of membrane formation). Warburg found that the hypertonic solution raises the rate of oxidat,ions in such eggs as much as ten times.j We repeated these experiments on the eggs of Strongylocentrotus purpuratus at Pa.cific Grove, and were able to confirm Warburg’s results although the rise in the rate of oxidations was much smaller than that observed in his experiments. In our experiments the rate of oxidations was first. determined for one and a half hours in normal sea water and then for one and a half hours in hypertonic sea water. Table V on the following page gives the results. The temperature was 18°C.

It should be pointed out that experiments 2 and 3, and 4 and 5, are made on equal lots of eggs. The results may serve as a check for the accuracy of the method.

The question arises: Why is it that the hypertonic solution causes a rise in the rate of oxidations in the unfertilized egg wit,h- out membrane formation, while it has no such effect on the same eggs after membrane formation ? The answer is that the hypertonic solution can cause the membrane formation and that it only raises the rate of oxidations in those eggs in which it causes membrane formation. In Loeb’s original method of causing artificial parthenogenesis by merely putting the eggs into a hypertonic solution, the hypert,onic solution had two kinds of effects: it caused first the membrane formation (or the formation of a gelatinous surface film) and at the same time furnished the curative effect.

5 TTarburg: Zeilsclc~. J. physiol. Chem., lx, p. 443, 1909.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


1

2

3

4

5

6

7

8

9

10

11

Normalseawater .................. Hypertonic (50 cc. sea water + 4 cc.

$- NaCl). .......................


$ NaCl). ....................... Normalseawater .................. Hypertonic (50 cc. sea water + 6 cc.

G NaCl). .......................

Normal seawater .................. Hypertonic (50 cc. sea water + 6 cc.

T NaCl). .......................

Normal seawater .................. Hypertonic (50 cc. sea water + 8 cc.

T NaCl). .......................


>$ NaCl). ....................... Normal sea’water .................. Hypertonic (50 cc. sea water + 9 cc.

T NaCl). ....................... Normalseawater .................. Hypertonic (50 cc. sea water + 9 cc.

-$ NaCl). .......................


?gM-- NaCl). .......................

Normalseawater .................. Hypertonic (50 cc. sea water + 12

CC. q NaCl) ....................

Normalseawater .................. Hypertonic (50 cc. sea water + 16

CC. 2; NaCl) ....................

mgm.

0.45 1.00

0.65 1.40

0.50 1.00

0.92

0.49

1.80

1.00

0.84 1.70

0.33 1.00

0.73

0.35

2.2Q

1.00

0.73 2.50

0.46 1.00

1.19 2.60

0.48 1.00

1.23 2.60

0.29 1.00

0.90

0.30

3.10

1.00

0.67 2.20

0.56 1.00

1.29 2.30

0.38 1.00

0.97 2.60


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


That the hypertonic solution can cause a cytolytic effect upon the cortical layer of the egg was shown by experiments on the eggs of Lottia (a mollusc) whose chorion can be liquefied by hypertonic solutions as well as by bases, if free oxygen is present.‘j

That the two agencies, the membrane-forming and the corrective one, can act simultaneously is not surprising. The same fact can be shown for bases. It is immaterial whether the base is applied first and is then followed by the corrective action of, the hypertonic solution or whether both agencies are applied simultaneously.

In our experiments the effect of the hypertonic solution upon oxidations was smaller than that caused by membrane formation with butyric acid. In the latter case, the rate of oxidations was raised from four to six times, while the hypertonic solution, when acting upon unfertilized eggs without membrane formation, raised the rate as a rule to not more than two and one-half and at the utmost three times the amount observed in the same eggs in normal sea water. This difference can be accounted for either by the fact that an exposure of one and a half hours of the eggs of S. purpuratus to hypertonic sea water as a rule does not suffice to bring about membrane formation and development; or by the fact that these experiments were made early in the season when fertilization did not raise the rate of oxidations as much as it did later in the season.

We repeated these experiments later in the season to find out whether the raje of oxidations would increase if the eggs remained a longer time in the hypertonic sea water. The temperature was 18°C. The consumption of oxygen was determined for each hour.

TABLE VI. I

UNFERTILIZED Em38 IN ; OXYOEN / CONBUMED

Kormal sea water.. . . . Hypertonic sea water; 1st hour.. Hypertonic sea water; 2d hour.. . Hypertonic sea water; 3d hour.. . Hypertonic sea water; 4th hour.. Hypertonic sea water; 5th hour..

Wm.

...... 0.16

....... 0.6i

...... 0.79 ...... 0.64 ...... 0.56 ...... 0.51

1.00 4.18 4.94 4.00 3.55 3.18

0 Loeb: Univ. of Calif. Publ. Physiol., iii, 1905.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

J. Loeb a’nd H. Wasteneys 477

A repetition of the experiment gave a similar result. In t.his case the hypertonic solution had the same effect upon the rate of oxidations as fertilization or membrane formation. It may also be that by chance we are dealing in this case with the eggs of a female which were very susceptible to the treatment with hypertonic solution while in the previous experiments the eggs had been more or less refractory. As we stated, not the eggs of every female of purpuratus develop if treated with hypertonic sea water.

These results support also the idea that the rise in the rate of oxidations is due to the membrane-forming action of the hypertonic solution, which of course takes place during the first two hours. We see that the maximal increase in the rate of oxidations takes place during that time.

IV.

If it is true that the increase in the rate of oxidations observed in these cases is merely due to the membrane-forming effect of the hypertonic solution, we should expect that if we add a weak base to the hypertonic solution, the increase in the rate of oxidations should be no greater than that caused by the weak base alone, the reason being that the weak base alone causes the membrane formation.7 This reasoning is supported by facts. The rate of oxidations was compared in unfertilized eggs (without membranes) in alkaline sea water and hypertonic sea water to which the same amount of base had been added. The hypertonic sea water consisted of 50 cc. sea water + 8 cc. -“g NaCl + KC1 + CaC12. The eggs were distributed into equal portions. One-half was put into 50 cc. of sea water + 1 cc. of the base; the other half of the eggs was first put into normal sea water and then into 50 cc. of hypertonic sea water + 1 cc. of the same base. Time of exposure one and a half hours; temperature 18°C.

7 Loeb: Journ. of Exp. Zodogy, xiii, p. 577, 1912.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


TABLE VII.

Normalseawater..................I 50 cc. hypertonic sea water + 1 cc.

G NEL~H . . . . . . . .I 50 cc. normal sea water + 1 x. g

NHtOH.. . . . . . Normal sea water. 50 cc. hypertonic sea water + 1 cc.

s benzylamine 50 cc. normal sea water + 1 cc. +$

benzylamine. Norma1 sea water................. 50 cc. hypertonic sea water + 1 cc.

& butylamine . 50 cc. normal sea water + 1 cc. $

butylamine......................

1.20 5.40

0.88 ! 4.00 0.37 1.00

1.89 / 5.10

1.75 / 4.70 0.36 [ 1.00

I I.73 / 4.80

1.67 1 4.60

It is obvious that the weak base alone raised the rate of oxidn- Cons practically to the same height as the combination of base and hypertonic sea water. The whole rise was due to the membrane- forming effect for which the weak base was sufficient.

In the case of a strong base, the result may be different, since neither the base nor the hypertonic solution alone may cause membrane formation (or the change in the cortical layer of the’egg) necessary for development. The following may serve as an example. Duration of experiment one and a half hours; t,em- pcrature 18°C.

TABLE VIII.

Iiormalseawater............................... 0.41 1 1.00 50 cc. hypertonic sea water + 1 cc. $$ NaOH 0.81 2.00 50 cc. normal sea water + 1 cc. 6 NaOH : ~ 0.46 1.20

In this case the NaOH had little effect and hence the hypertonicity caused a noticeable increase in the rate since it probably increased the number of eggs in which the process of membrane formation was started.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Finally, we wish to report an experiment which does not strictly belong here but which shows that the mere cytolysis of the cortical layer is responsible for the increase in the rate of oxidations observed after membrane formation either by a spermatozoon or by butyric acid. It is possible to cause complete cytolysis of the unfertilized egg of X. purpuratus with saponin and we found that this increases the rate of oxidations to the same extent as fertilization by sperm. The following may serve as an example. Gnfer- tilized eggs of S. purpuratus were used; temperakrre 15’C.

TABLE IX.

Unfertilized eggs. _. .~ 0.15 1.00 The same eggs after cytolysis with saponin. .’ Unfertilized eggs. _’

1.07 ~ 7.10 0.22 ~ 1.00

The same eggs after cytolysis with saponin.. 1 0.80 .I 3.60

The variation in the effect of cytolysis in the two experiments may be due to the fact that in the second experiment an exces- sivc amount of saponin was used.

This experiment proves that the increase in the rate of oxidations due to fertilization or artificial membrane formation is merely caused by the cytolysis of t.he corOica1 layer.

VI. THEORETICAL REhl4RKS.

It seems that all the experiments point very clearly towards one conclusion, namely, that the hypertonic solution raises the rate of oxidations in the unfertilized eggs of X. purpuratus only under one condition, i.e., if it causes the change at the surface of the egg underlying membrane formation. In eggs which have undergone 6he process of membrane formation either by fertilization or by a treatment with butyric acid, the hypertonic solution causes no further increase in the rate of oxidations.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


SUMMARY.

1. The unfertilized eggs of sea urchins which have undergone artificial membrane formation die if not treated with a hypertonic solution. It is shown in these experiments that the rate of bxida- tions in such eggs is not increased by the hypertonic solution.

2. It is shown that the hypertonic solution does not cause an increase in the rate of oxidations of fertilized eggs of Strongylo- centrotus purpuratus.

3. Hypertonic solutions increase the rate of oxidations in unfertilized eggs which have not undergone the process of membrane formation, as Warburg observed. This increase is purely due to the fact that the hypertonic solution induces the change in the cortical layer of the egg which leads to membrane formation.

4. This conclusion is supported by the fact shown in this paper, that the addition of a weak base to normal sea water, which causes development (or membrane formation) increases the rate of oxidations in unfertilized eggs to the same amount as if it were added to hypertonic sea water. Since in this case the membrane-forming effect can be produced by the base alone the addition of the hypertonic solution can add nothing to the effect.

5. Complete cytolysis of the unfertilized egg by saponin raises the rate of oxidations to the same height as fertilization, thus showing that the cytolysis of the cortical layer of the egg is the essential feature in fertilization.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Jacques Loeb and Hardolph WasteneysUNFERTILIZED EGGS

OXIDATIONS IN FERTILIZED ANDSOLUTION UPON THE RATE OF

THE INFLUENCE OF HYPERTONIC

1913, 14:469-480.J. Biol. Chem.

http://www.jbc.org/content/14/5/469.citation

Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

alerts to choose from all of JBC's e-mailClick here

ml#ref-list-1

http://www.jbc.org/content/14/5/469.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be by guest on February 15, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/14/5/469.citation

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;14/5/469&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/14/5/469.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=14/5/469&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/14/5/469.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/14/5/469.citation.full.html#ref-list-1

http://www.jbc.org/content/14/5/469.citation.full.html#ref-list-1

http://www.jbc.org/

the influence of hypertonic solution upon · the influence of hypertonic solution upon the rate of...

Documents