Vapour Pressure Changes in the Frog’s Egg at Fertilization

The suggestion has been made that the response of an egg to the action of a spermatozoon or parthenogenetic agent may be similar to that of a nerve to stimulation (Lillie, 1909). If this were so, it might be expected that activation, whether by a parthenogenetic agent or by a spermatozoon, would cause an increase in the permeability of the egg surface ; although this increase in permeability is transient in the case of stimulated nerve, muscle, or Nitella cells, it need not necessarily be so. On the other hand, though the plasma membrane of the sea-urchin egg is known to become more permeable to sparingly ionized substances after fertilization (Lillie, 1917), there is no evidence that it is more permeable to ions, as would be expected if activation were strictly analogous to the stimulation of nerve or muscle. There is, in fact, some evidence to the contrary; the most recent investigations make it doubtful if any decrease in membrane resistance after fertilization has ever been measured in sea-urchin eggs (Cole, 1928).

In the frog’s egg, which, like that of the sea-urchin, has been studied intensively, profound changes in osmotic pressure were said by Backmann & Runnström (1912, p. 344) to take place after fertilization : ‘Durch die Befruchtung wird in der Eizelle von Rana temporaria eine erhebliche Reduktion des osmotischen Druckes des Ei-inhaltes hervorgerufen. Der osmotische Druck wird etwa bis zu einem Zehntel des osmotischen Druckes beim erwachsenen Frosch reduziert. Es herrscht Isotonie zwischen dem eben befruchteten Froschei und dem umgebenden Wasser.’^*

Their results are displayed in Table 1. From these it would seem that the fall in osmotic pressure in unfertilized eggs in tap water is much less than in fertilized eggs soon after fertilization; even after 36 hr. the fall in osmotic pressure in unfertilized eggs in tap water is not as great as in fertilized eggs.

The interpretation of the results given in the text of Backmann and Runnström’s paper, however, seems to differ from that expressed in the summary already quoted. In the text it is suggested that the fall in osmotic pressure is due not to fertilization, but to the hypotonicity of the pond water, which progressively cytolyses the eggs in the absence of the stabilizing or corrective effect of fertilization or parthenogenetic activation.

Further work by other experimenters in this field (Backmann & Sundberg, 1912; Bialaszewicz, 1912;Przyleçki, 1917; Voss, 1920;Krogh, Schmidt-Nielsen & Zeuthen, 1938) has failed to make clear the relation between osmotic pressure changes and fertilization, and it therefore seemed advisable to repeat Backmann & Runnström’s experiments, measuring vapour pressures rather than freezing-point depressions, in order to establish what change in vapour pressure occurs in the frog’s egg on fertilization, and in the unfertilized egg on transference to tap water.

The eggs of the common frog, R. temporaria, were used. In some experiments measurements were made on unfertilized, ‘uterine ‘eggs, while in others ‘coelomic ‘eggs were used. The latter are eggs which have left the ovaries but have not as yet entered the oviducts. Coelomic eggs are very convenient material since they lack a covering of jelly.

The method of obtaining high percentages of fertilized eggs was as follows. A frog was pithed, and the eggs from the two ‘uteri ‘were spread in a layer one egg thick over the bottom of two Petri dishes, about 10 cm. in diameter. A third Petri dish of the same size was half-filled with tap water and two testes and two seminal vesicles were placed in it. These were then crushed, and the contents of the dish stirred to give a homogeneous suspension of sperm. This was poured over the eggs in one Petri dish, and left for 10 min. Simultaneously, a similar volume of tap water was poured over the control eggs in the other dish. After 10 min. the two dishes were placed separately in large volumes of tap water. In general a high percentage of eggs was fertilized and cleaved normally; no control eggs cleaved. As Cambridge tap water was not at that time (1935) of constant composition, artificial media of standard composition were sometimes used. Cleavage and development were normal in these.

‘Uterine’ eggs are covered with a dense layer of jelly, which cannot be dissected off without damaging the eggs. They are also surrounded by a membrane (between egg and jelly), often known as the vitelline membrane, but which, in accordance with the nomenclature accepted for the trout egg, it is here proposed to call the chorion. When the unfertilized frog’s egg is placed in tap water, the chorion becomes thicker and tougher, as in the trout egg. After being in water for some time, a space appears between the chorion and cell surface; this is the perivitelline space and is filled with fluid. When it appears, the egg becomes free to rotate, until its centre of gravity is in the equilibrium position, and sinks to the bottom of the perivitelline space. If an egg with a well-defined white pole is now turned upside down, it rotates until the white pole, towards which the centre of gravity lies, once more faces vertically downwards. Here there is a parallel in behaviour between the frog’s egg and the trout egg.^*

When an egg is fertilized the same phenomena are observed, but the perivitelline space appears more rapidly than in the unfertilized egg. After eggs have been successfully inseminated, they will rotate in about 20 min. ; in the case of unfertilized eggs, a period of $112$–3 hr. may elapse before rotation occurs.

The vapour pressure of the contents of fertilized and unfertilized eggs was determined by the differential thermal method of Hill (1930), which has advantages over the Beckmann freezing-point depression technique used by Backmann & Runnström and most other experimenters in this field. The instrument for determining vapour pressures was similar to that described by Hill (1930) and Margaria (1930). Two thermopiles were used for each determination, and their two readings were averaged. A number of determinations were made in an atmosphere of nitrogen to see if the heat production of material obtained by squashing the eggs caused any appreciable error. The results were negative. All manipulations were made in a small moist chamber (Picken, 1936).

As a preliminary experiment, and as a check on the method, the vapour pressure of frog’s blood was determined. Ten pairs of determinations were made, and the results, expressed as decimal fractions of the vapour pressure of a 1 % NaCl solution, are shown in Table 2. It is clear that there is considerable variation between one frog and another, and this has to be taken into consideration in all experiments described in this paper. The values for the vapour pressure of the eggs of different females also vary considerably from one female to another, but are closely correlated with the values for the vapour pressure of the blood from the same females (Fig. 1).

Results of measurements on unfertilized ‘coelomic’ eggs and on ripe ‘uterine’ eggs are shown in Table 3. In these experiments the material obtained by squashing five eggs, or alternatively by sucking up the contents of five eggs in a fine glass pipette, was placed on a filter-paper applied to one face of a thermopile. A 1 % solution of NaCl was applied to the filter-paper on the other face of the thermopile.

The main experiments on fertilized and unfertilized eggs were carried out after the eggs had been in water for half an hour, during which time the jelly swells. Five eggs were selected, and the jelly surrounding each was carefully dissected off with micro-scissors, leaving the eggs bare except for the chorion, which was not removed. The eggs were then ground up, and the pulp was placed on a filter-paper on the face of a thermopile.

The experiments fall into two groups: first, measurements of vapour pressure made half an hour after insemination; and secondly, measurements made 3 hr. after insemination. All fertilized eggs cleaved within 3 hr., but no fertilized eggs cleaved within half an hour of insemination; difficulties therefore arose in selecting unquestionably fertilized eggs from the fertilized batch, half an hour after insemination. At the beginning of the season, all ripe eggs have a well-defined animal and vegetative pole. As the fertilized eggs begin to rotate about 20 min. after insemination, it is possible, at the beginning of the season, to select fertilized eggs after half an hour by removing those which have clearly undergone rotation. All eggs, however, do not have a well-defined white pole, particularly when the breeding season is advanced. In these circumstances there is no visible difference between the animal and vegetative pole, and it is impossible to observe rotation, though the eggs cleave in the normal way. It was, therefore, thought inadvisable to select eggs which had rotated and were thus known to be fertilized, when in other experiments selection was necessarily random, since no rotation was visible. Had this selection been made, the results of different experiments (with and without selection) would not have been comparable. In the half-hour experiments, therefore, eggs from the inseminated batches were selected at random, no attention being paid to rotation or lack of rotation. After the selection had been made, the inseminated eggs were left in their Petri dish for 3 hr., by which time the first cleavage had taken place. Each dish held about 600 eggs. The total number of cleaved and uncleaved eggs was then counted. The results of these experiments are shown in Table 4.

The arithmetic means of these results suggest that the fall in vapour pressure in fertilized eggs is greater than in unfertilized eggs, though in both fertilized and unfertilized eggs in water the vapour pressure is lower than in unfertilized eggs before immersion in tap water. It is difficult to analyse these results statistically, because the eggs from different frogs must be considered as different populations. Individual results, however, show a distinct trend in the same sense as the arithmetic means.

A further series of determinations was made 3 hr. after insemination, that is, while the eggs were in the two-cell stage. At this period Backmann & Runnström obtained very low values for the osmotic pressure. They claimed that soon after fertilization the osmotic pressure of the egg contents was about the same as that of pond water, though the osmotic pressure of the unfertilized eggs after 3 hr. in water was considerably higher. The uncertainty in determinations made half an hour after insemination—that is, whether eggs were fertilized or not—did not arise in these experiments, since only eggs which had cleaved were used. The values recorded are shown in Table 5. One anomalous result was obtained, in which fertilized eggs had a vapour pressure of 0·69 and unfertilized eggs 0·55% NaCl. The difference in vapour pressure between fertilized and unfertilized eggs 3 hr. after insemination and immersion in water is not significant.

To test this result a few experiments were made with material from unfertilized eggs on one thermopile face and that from fertilized eggs on the other. These experiments confirmed the results given in Table 5 and are in serious disagreement with those of Backmann & Runnstrom. The extent of this disagreement can be seen in Fig. 2, where the values for the osmotic pressure of the eggs are compared graphically.

Some preliminary conductivity measurements were carried out on fertilized and unfertilized egg pulp. These experiments, which presented considerable technical difficulties, were made in collaboration with the late Oliver Gatty. The results suggest that after fertilization there is approximately 10% less electrolyte in the egg than before fertilization, which is in qualitative agreement with the results of McClendon (1915); but the experiments should be repeated before the results can be accepted

Our experiments show that 3 hr. after fertilization in tap water, or 3 hr. after transfer to tap water, there is no difference between the vapour pressure of fertilized and unfertilized eggs. In both, however, the vapour pressure has fallen to about 0·66 of its initial value. Backmann & Runnstrôm showed that the increase in volume of eggs after fertilization was at most 6·6% and calculated that a water uptake of this amount would lead to a fall in osmotic pressure from that of a 0·7 % solution of sodium chloride to that of a 0·58% solution, that is, a fall of about one-sixth. The fall observed by us would therefore be accounted for if the egg were semipermeable and swelled in tap water. This is in fact what Krogh et al. (1938), who also used the differential thermal method for measuring vapour pressures, believe to happen in the fertilized egg.

Preliminary conductivity experiments suggested, however, that electrolytes disappear from the egg after fertilization. That is to say, some of the fall in vapour pressure might be due to disappearance of electrolytes from the contents of the egg. The simplest hypothesis to account for this is that outward diffusion of ions into the external medium occurs, until a new dynamic equilibrium between egg and environment is established.

McClendon (1915) obtained similar results, but his experiments might be criticized on the grounds that the existence of a single dead egg in his suspensions of fertilized eggs would have vitiated his conclusions.

Backmann & Runnström pointed out, however, that fertilized eggs will develop in repeatedly changed distilled water, and argued from this observation that there can be no continuous loss of electrolytes by diffusion from the egg, since the salt content of the egg would soon be exhausted under these conditions, and development would be impossible. They concluded, since the swelling of the fertilized eggs was not sufficient to account for the reduction in osmotic pressure to about one-tenth of the initial value, that the number of osmotically active molecules and ions in the egg substance is reduced as a result of fertilization, not by water entering, nor by ions or molecules diffusing out, but by adsorption within the egg due to a change in the state of the cell colloids.

If, as our results show, there is no such precipitous fall in the vapour pressure of the fertilized egg to isotonicity with the surrounding tap water, Backmann & Runnström’s hypothesis is superfluous.

The discrepancy between our results and those of Backmann & Runnström is not easy to understand. The Beckmann method of determining freezing-point depressions is, for various reasons, not entirely satisfactory when dealing with highly viscous material of this kind. About i c.c. of fluid is needed for accurate determinations, and this would mean using a large number of eggs for each determination. But in order to remove the jelly completely, 5 or 10 min. may have to be allowed for the dissection of each egg. One source of error may, therefore, have been incomplete removal of the jelly, the presence of which would tend to dilute the egg contents and lead to a low value for the vapour pressure. It is unlikely, however, that errors introduced in this way could account for the discrepancy, which amounts to the difference between the osmotic pressure of frog’s blood and that of tap water. Moreover, the results of Backmann & Runnström’s determinations on frog’s blood and unfertilized eggs are similar to ours, and this again suggests that the discrepancy cannot only be due to the inadequacies of the Beckmann method.

In general agreement with our results, Cole & Guttman (1942) found no significant change in the resistivity of the frog’s egg ‘cytoplasm ‘nor in membrane resistance, after fertilization. Though they make no mention of chorion, perivitelline space or conductivity of the perivitelline fluid—all of which might affect the calculations of membrane resistance^*—it is hardly conceivable that their alternating current experiments should have failed to reveal a difference between the conductivity of fertilized and unfertilized egg contents, if Backmann & Runnström’s results are correct.

The small difference between the vapour pressure of fertilized and unfertilized frog’s eggs, half an hour after fertilization, could be accounted for by the more rapid diffusion of salts out of the egg, through the chorion and into the external medium, or of water into the egg, as a result of fertilization. If the salts diffused out of the egg and into the perivitelline space, but not through the chorion, vapour pressure measurements would only reveal a difference between fertilized and unfertilized eggs if water entered the perivitelline space, since the chorion was not dissected away from the eggs.

As unfertilized eggs develop a perivitelline space after immersion in tap water, the idea that the extrusion of the chorion is a direct result of fertilization must be rejected. It is true that the perivitelline space appears much more quickly in fertilized eggs ; but the fact that it can appear, albeit more slowly, without fertilization, after the eggs have been transferred from the ‘uterus ‘to a hypotonic medium, suggests that this transfer is the responsible factor. This was pointed out by Przyleçki (1917). The spermatozoon appears to alter the rate at which the perivitelline space is formed, or the rate at which a new dynamic equilibrium between the egg and its environment is established. This is confirmed by the experiments of Voss (1926).

The appearance of a perivitelline space in unfertilized eggs in tap water suggests that the hypotonicity of this medium acts as an abortive parthenogenetic stimulus. The spermatozoon seems to be more efficient than the hypotonic medium in causing the perivitelline space to appear ; at the same time, perhaps as a result of a change in the cell surface, there is slightly less tendency for osmotic equilibrium to be approached between the egg and its environment than in the fertilized egg.

Thus far there has been no discussion of the mechanism by which the perivitelline space is produced. Its formation may be an osmotic phenomenon due to the liberation of substances of high molecular weight into the space between the chorion and the vitelline membrane (Bialaszewicz, 1912); the elevation of the fertilization membrane in sea-urchin eggs is probably due to a reaction of this type (Loeb, 1908). In unfertilized eggs we may suppose that these substances diffuse out more slowly.

Unfertilized frog’s eggs swell more than do fertilized eggs in tap water, and in them the fall in vapour pressure seems to be the first step in cytolysis. They do not survive for any length of time in tap water, unlike unfertilized trout eggs, which can be kept in a healthy condition for weeks in this medium. This difference is probably due to the impermeability of the vitelline membrane of the trout egg.

If the analysis of the vapour pressure changes is to be carried further, we require simultaneous measurements of volume changes, vapour pressure changes in the eggs, and electrolyte concentration changes in the eggs and in the surrounding medium. At the moment it seems reasonable to conclude that, if Backmann & Runn-ström’s observations on volume changes are correct, these would more than account for the fall in vapour pressure observed in fertilized and unfertilized eggs, supposing that the increase in volume is due to the uptake of water.

Concerning the postulated change in permeability of the egg surface on fertilization, the vapour pressure experiments provide neither evidence for nor against such a change. They show only that the fall in vapour pressure is of the same order as that which might occur as a result of the volume change. The fact that the contents of the egg do not become isotonic with the surrounding tap water indicates that at some stage after the egg, whether fertilized or unfertilized, is transferred to tap water, the exchange of water and dissolved substances between egg and surrounding medium is actively controlled.