Cytolysis and Protoplasmic Structure: III.—Antagonism of Hypotonic Cytolysis by Saponin in the Starfish Ovum

The experimental method employed was as follows: approximately 20,000 eggs contained in ¼ c.c. of sea-water were introduced into a series of watch glasses containing the various solutions. At definite time intervals comparable amounts were removed for washing, subsequent chemical treatment and microscopic examination. The time of complete cytolysis in hypotonic solutions was employed as an index of the protection afforded the eggs by saponin. Hypotonic solutions were made by mixing sea-water and tap-water to the required proportions; for example, 70 parts of tap-water with 30 parts of sea-water gives the so-called 70 to 30 mixture.

In order that we may have an index of the viability of the eggs after saponin and hypotonic treatments, it seemed advisable to repeat some of the above experiments with fertilised eggs and determine their ability to develop subsequently in sea-water. Table III. gives a summary of the results of at least five experiments at each concentration.

Since eggs previously treated with saponin are more resistant to hypotonic solutions than when not so treated, it might be anticipated that an even greater protective effect would be obtained by introducing the eggs into a hypotonic solution in which the saponin had previously been dissolved. This was found to be the case.

1. A 70 to 30 hypotonic solution containing 3 per cent, saponin caused cytolysis of immature Asterias eggs only after fifteen minutes.

2. A 70 to 30 hypotonic solution containing 10 per cent, saponin was used to treat fertilised Asterias eggs for four minutes. It was found that development proceeded quite normally on subsequent washing and returning in sea-water.

Fertilised eggs were treated with 70 to 30 hypotonic solution for periods not in excess of two minutes, then with 15 per cent, saponin for four minutes followed by washing in sea-water. These eggs seemed to develop normally in the same number and with the same velocity as the controls. This number was 30 per cent, in excess of the number of developing eggs that had been treated with the hypotonic solution for two minutes without subsequent treatment with saponin. However, a longer preliminary hypotonic treatment caused cytolysis which obviously could not be reversed by subsequent saponin treatment.

Since it appeared a reasonable assumption that the surface film of the egg adsorbed the saponin to form a protective film, eggs were treated for one minute with 1 to 3 dilution of a saturated sea-water solution of ethyl ether. These eggs were then placed in 15 per cent, saponin for three minutes, washed for two minutes, and finally placed in 70 to 30 hypotonic solution until they were completely cytolysed.

From the results presented in Table IV. it is evident that while a brief treatment with ether of this concentration considerably increases the resistance of the egg to hypotonic solutions, a treatment with ether followed by saponin renders the egg more susceptible to hypotonic than is the normal egg. The increased susceptibility to hypotonic of eggs previously treated with ether for the recorded time and concentration, followed by saponin, suggests the possibility that the ether alters in some manner that portion of the protoplasmic interfaces which is able to combine with the Quillaja saponin to protect it from hypotonic cytolysis.

The viscous jelly may be removed from the egg by the method adopted by Ralph Lillie²  which consists of shaking the eggs with 0.54 M NaCl. Also eggs may be entirely deprived of their investing jelly by means of the microdissection needle. Eggs prepared by either of these methods exhibited identically the same resistance to hypotonic solutions after saponin treatment as did eggs which had not been deprived of their jelly.

The egg membrane has long been known to be freely permeable to electrolytes. To test its permeability to saponin fertilised eggs were introduced into a 30 per cent, saponin solution in sea-water and it was noted that no shrinkage of the egg membrane occurred. In addition, by using a micropipette of less than 1/2 µ µ in diameter, saponin was injected immediately under the egg membrane of a mature egg. A bulging of the membrane occurred, quickly followed by a collapse of the bulge, the egg then appearing normal. The same effect was obtained with the fertilised egg, but in this case either due to the liquefaction of the protoplasm on fertilisation, or to the increased toughness of the egg membrane, the injected saponin droplet formed a depression in the egg. Very soon this concavity was lost and the egg rounded up to its normal size and shape, demonstrating that the saponin had diffused through the freely permeable fertilisation membrane. That the saponin probably did not diffuse into the protoplasm is suggested by the fact that no viscosity or volume changes could be detected with the method employed.

From these experiments it may be concluded that saponin passes freely through the membrane, and it appears extremely improbable that either the membrane or the surrounding jelly play any primary rôle in the saponin hypotonic antagonistic effects.

Dr Robert Chambers has carried out, in conjunction with the authors, microdissection studies in order to demonstrate physical and morphological changes incident to saponin treatment.

Briefly, it has been found that—

• (1) Spherules of artificially extruded endoplasm exhibit the saponin-hypotonic antagonism but not to the same degree as intact eggs.

• (2) The cortical portion of the egg, which remains after the extrusion of most of the endoplasm, was not as resistant to hypotonic cytolysis after saponin treatment as was the endoplasmic sphere or the whole egg. The same has been found true for hypotonic and digitonin solutions. (Page, Chambers, and Clowes.³ )

• (3) Saponin markedly toughens the membrane of the Asterias egg. Inasmuch as quite normal endoplasmic spheres may be produced from saponin treated eggs we recognise the importance of surface concentration in this antagonism.

• (4) Saponin may be injected into the egg without causing cytolysis, provided time is allowed for the formation of a surface film. If the speed of injection does not allow of the mobilisation and organisation of this protection cytolysis follows rapidly.

1. Quillaja saponin has the power in the starfish egg under certain conditions recorded in this paper of antagonising the cytolytic action of hypotonic solutions.

2. A differential protection of a protoplasmic moiety susceptible to hypotonic solutions has been demonstrated, as shown by the following observations.

   • (a)

     Starfish eggs are more resistant to hypotonic cytolysis when given a preliminary treatment with saponin. Within limits the eggs are still viable after the saponin-hypotonic treatment.

   • (b)

     The artificially extruded endoplasm shows the saponin-hypotonic antagonism, although the cortical proto-plasm remaining after the extrusion was rendered less resistant to hypotonic cytolysis after saponin treatment.

   • (c)

     Saponin dissolved in a hypotonic solution renders that solution much less cytolytic than the control containing no saponin.

   • (d)

     Preliminary treatment with ether renders the saponin treated eggs much more susceptible to hypotonic cytolysis than the control in which no ether was used.

   • (e)

     The investing jelly and egg membrane are not primarily important in the antagonism.

   • (f)

     Saponin may be injected into the egg without causing cytolysis, provided time is allowed for the formation of a surface film.