1. Embryos of Siredon mexicanum and Xenopus laevis were shown, by the use of 32P, to take up small but measurable amounts of phosphate from the culture medium.

  2. The rate of uptake was higher in embryos than in unfertilized eggs, and was related to the stage of development. The rate was proportional to the concentration of phosphate in the medium.

  3. Spontaneously occurring microcéphalie embryos took up much more phosphate than normal embryos.

  4. Injured embryos and dissected fragments of embryos took up more phosphate than intact embryos. Dissected neurulae took up more phosphate than dissected blastulae, and half-gastrulae took up more than either.

  5. Vegetative fragments of blastulae took up more phosphate than animal fragments, particularly towards the end of the 6-hour period studied.

  6. Ventral fragments of gastrulae took up the same amount of phosphate as dorsal halves during the first 2 hours after cutting, but subsequently took up more.

  7. The phosphate uptake of dorsal fragments of neurulae was at first the same as that of ventral fragments, but from 4 to 6 hours after cutting was about twice as rapid.

  8. 10 – 4 M p-dinitrophenol had a slight inhibiting effect on uptake of phosphate by whole embryos, counterbalanced by adsorption of 32P on killed cells. The phosphate uptake of fragments was cut down by the inhibitor to about one-half to one-third the level of the controls.

THIS work on the introduction of radioactive phosphate into amphibian embryos was carried out as a necessary preliminary to research on embryonic phosphate metabolism, in which 32P was to be used as tracer. It has an interest of its own for two reasons.

In the first place, recent work on the penetration of phosphate into cells in general suggests that this phenomenon is not simply a result of diffusion, but is related to the metabolism of the cell (cf. Danielli, 1954), and may be mediated by a ‘carrier’ at the cell membrane (cf. Sutcliffe, 1954, and Russell, 1954, with reference to plant cells, and Sacks, 1948, and Popjak, 1950, for mammalian cells). If these hypotheses are confirmed, differences in phosphate uptake which may appear among embryonic cells in the course of development may be regarded as indicative of metabolic differences, and even possibly of differentiation of membrane structure. The importance which such a membrane differentiation might have in development has been presented in abstract terms by Weiss (1953). Therefore, in spite of the fact that amphibian embryos do not, in general, need phosphate from an outside source for their development, if different stages and different types of cells show differential permeability to the ion, this may indicate interesting metabolic differences.

In the second place, results for Amphibia may be compared with the extensive data available on the subject of phosphate uptake by echinoderm embryos. Marine embryos such as echinoderms normally absorb considerable quantities of phosphate from sea-water during their development. This phosphate absorption, which is a necessary function of the embryo, is very sensitive to such factors as change of temperature and the presence of metabolic inhibitors (cf. Discussion). It is interesting to contrast the behaviour of these embryos with that of amphibian embryos, which have evolved a relative independence of their environment.

So far as Amphibia are concerned, little work has been published. Villee & Duryee (1951) kept unfertilized eggs of Triturus pyrrhogaster in a medium containing 1 μC. per ml. of 32P, and after 6–24 hours were able to fractionate the labelled compounds chemically. Hansborough & Denny (1950,1951) found that phosphate penetrated embryos of Rana pipiens. Measurements of the penetration rates were not given in either of these papers. Kutsky (1950) reported only a negligible penetration of phosphate into R. pipiens embryos without giving details.

Eggs

The axolotl eggs used in the earlier experiments were obtained in spring by bringing together male and female animals previously kept apart. The eggs of Xenopus laevis were all obtained by injection of the parents with ‘Pregnyl’ (chorionic gonadotropin).

Isotope

The 32P was supplied as sterile, carrier-free, radioactive orthophosphate in a small volume of dilute hydrochloric acid.

Media

Radioactive culture media were made by adding the isotope to Holt-freter solution which had been boiled and cooled. This medium was then neutralized with sodium bicarbonate. If whole embryos were to be incubated, one-third-strength Holtfreter solution was used; if dissected parts of embryos, full-strength solution. When dinitrophenol was used, it was added before boiling the solution.

Concentrations of from 5 to 10 μC. 32P per ml. of solution were used in these experiments. In each case samples of the medium were evaporated on planchettes and the content of 32P was estimated with an end-window Geiger-Muller counter.

The ‘carrier-free’ 32P contains a very small quantity of 3 IP, which varies from one batch to another. The total 31P + 32P is too small to estimate chemically. The specific activity (i.e. counts per minute of 32P per mg. phosphate) of the media could therefore only be standardized by dilution with a known, larger quantity of 3 IP. The number of counts per minute per unit weight of phosphate could then be stated, and the actual amount of phosphate taken up by the embryos could be calculated. However, as the embryos take up so little phosphate, it seemed advisable to add the isotope mixture to a perfectly phosphate-free solution, sacrificing the possibility of knowing the exact amount of phosphate which had entered the cells to the retention of the very high specific activity of the initial ‘carrier-free’ solution.

The concentrations and uptake of phosphate are therefore expressed in counts per minute per millilitre of solution or per embryo.

Experiments

Before the embryos were incubated in the radioactive medium, the jelly was removed with forceps, and they were washed by transference through four dishes of sterile one-third-strength Holtfreter solution.

Dissections of embryos were performed with the point of the forceps in full strength Holtfreter solution on a layer of agar. When the whole group had been cut up as quickly as possible 10 minutes were allowed to elapse before the fragments were transferred either to the 32P medium or to a larger agar-lined dish to await later incubation. The first phase of closure of the wound was therefore completed before the fragments were moved.

The temperature was kept constant during the whole of any one experiment, but was not the same for all experiments.

At the end of the incubation in the 32P medium the embryos or fragments were pipetted into a large volume of sterile Holtfreter solution, and washed by repeated transferences until the washing medium was no longer radioactive. This usually took from 10 to 15 minutes.

Fragments of embryos were then pipetted individually on to planchettes and dried for estimation of 32P by means of an end-window Geiger-Muller counter.

Early experiments with axolotl embryos showed that their vitelline membranes do not become radioactive, so that there was no need to remove these before drying the embryos. The vitelline membranes of Xenopus embryos usually have adhering strands of jelly, and become extremely radioactive in a medium containing 32P. Before the whole Xenopus embryos were dried, therefore, the membrane was removed. This operation was usually delayed for a few hours for the sake of convenience. It was found that there was no measurable loss of the 32P once it had been taken up.

The uptake of 32P was so small that it would have been almost impossible to estimate the 32P content of each embryo individually. Groups of from two to six were therefore dried together on each planchette. These formed so very fine a film of material that it was not necessary to correct the results for self-absorption of electrons.

Two counts were taken of each sample, each of these being high enough by itself to keep the counting error below 3 per cent. The counter was checked daily with a uranium sample. All counts were corrected for radioactive decay.

Cytological technique

The following was found most satisfactory for dissected parts and for whole embryos.

Fixation overnight in 20 per cent, trichloracetic acid at about 2° C. (in the refrigerator). Dehydration in 70 per cent, ethanol at 2° C. after a rinse in distilled water at the same temperature; then allowed to rise to room temperature, transferred after 1 hour to 90 per cent, ethanol and after a further hour to 100 per cent, ethanol. After a total of 2 – 3 hours in 100 per cent, ethanol (two changes) cleared in chloroform for 2 – 3 hours (or overnight). Embedding in wax in the usual way, with not more than two changes each of three-quarters of an hour in hot wax. Some whole embryos, including samples of those treated with dinitrophenol, were embedded and sectioned in the same way.

Controls

The possibility of radiation damage to the embryos was carefully checked. Embryos which had been incubated in the radioactive medium were compared with two kinds of controls, A and B.

A. Random groups of the same batch of eggs were kept in open dishes in tapwater. Among these embryos many hatched, some were reared to metamorphosis, and occasionally a few were reared to maturity. Some were infertile or had suffered damage by the parent toads, and in some spontaneous anomalies such as microcephaly and exogastrulation occurred. Mortality was fairly high.

B. A group of embryos was dejellied, washed, and cultured in sterile conditions in one-third-strength Holtfreter solution. As in the experimental group, all defective or abnormal embryos were carefully excluded at the time of selection.

The frequency of abnormality and mortality was the same in the control group B as in the experimental groups (usually about 1 to 2 per cent.) and was much lower than in control group A.

Embryos which had been kept in a radioactive medium for a long time did not show a greater liability to defect than those incubated for a shorter time. The rate of development of irradiated embryos was the same as that of the controls. No signs of radiation damage were seen in sections of embryos and fragments incubated in 32P.

The uptake of phosphate by whole embryos at different stages of development

In preliminary experiments embryos of Siredon mexicanum were incubated continuously in radioactive medium from the uncleaved egg stage until the tail-bud stage, and a number of embryos was taken out at each stage for estimation of the 32P taken up. This technique gave substantially the same results as subsequent experiments with short periods of incubation, but was unsatisfactory in the first place because of the prolonged irradiation, and in the second because bacteria eventually invaded the medium. Towards the end of the experiments the embryos showed signs of cytolysis, and by shedding cells actually lost radioactivity. Several unfertilized eggs which were included in these experiments showed no signs of cytolysis throughout the long period in radioactive medium. In contrast to the developing embryos they were almost completely impermeable to phosphate. For instance, in one experiment, after 46 h. 20 min. in the radioactive medium, three mid-gastrulae had taken up 423,448, and 509 counts per minute of 32P respectively, three unfertilized eggs only 15,10, and 20 counts per minute respectively.

Two experiments with 1-hour incubations in radioactive medium were carried out. There were inexplicable differences between the two sets of results, but in both it appeared that there was a rather higher uptake during the cell movements of gastrulation and neurulation, followed by a definite fall in penetration rate after closure of the neural folds. An experiment with a single batch of embryos of Triturus alpestris gave similar results.

These first experiments were carried out in the laboratory of Professor J. Brachet of Brussels University. Subsequent experiments were carried out in London with a different stock of Siredon mexicanum. The embryos produced by this stock proved to be totally impermeable to radioactive phosphate.

Embryos of Xenopus laevis were therefore used for further experiments. Results are given in Table 1. These embryos develop so quickly that the 2-hour incubations cover almost the whole period of development from cleavage to formation of the tail-bud. For instance, the mid-gastrula (yolk-plug) stage has reached the early neurula stage at the end of the incubation period. The numbers in each group as given in the table represent numbers of radioactive samples counted; each sample consisted of several embryos.

TABLE 1

The uptake of phosphate by whole embryos of Xenopus laevis expressed in mean counts per minute of 32P per embryo taken up in 2 hours with standard error

The uptake of phosphate by whole embryos of Xenopus laevis expressed in mean counts per minute of 32P per embryo taken up in 2 hours with standard error
The uptake of phosphate by whole embryos of Xenopus laevis expressed in mean counts per minute of 32P per embryo taken up in 2 hours with standard error

Since a different sample of 32P was used for each experiment and each sample has a different specific activity, the absolute values are not comparable from one experiment to the next. The variation between different experiments is high, which is understandable in view of the minute quantities of material involved, but the relative values for different stages show the same sort of pattern in the various experiments. The gastrula and neurula stages in general have the highest uptake, the tail-bud stage always takes up least phosphate.

The penetration of phosphate into unfertilized and uncleaved but fertile eggs could not be measured, since it is almost impossible to remove the jelly from these without causing severe injury.

In most experiments one dish of medium was used for all the stages. The uptake was so slight compared with the concentration in the medium that depletion of the 32P was negligible, but it seemed possible that the results might be falsified by growth of bacteria. In two experiments, therefore, a fresh dish of medium was used for each stage for one of three groups (see Table 1). There was no obvious difference in the group so treated. The estimated radioactivity of the various media was as follows: experiment 1: 554,000 counts per min. per ml.; experiment 2: 1,338,000 c./m.; experiment 3: 1,474,000 c./m.; experiment 4: 1,584,000 c./m.; experiment 5:1,390,000 c./m. (approx. 10 μ C. per ml. in the last three).

Abnormal embryos

Two series of Xenopus embryos were incubated in the radioactive medium throughout development, and samples were taken at intervals as in the preliminary axolotl experiments. There was no cytolysis or appearance of infection in these, but the results were unsatisfactory in that variability in uptake became progressively greater at late stages. The interest of these experiments is that among the embryos, all of which had been apparently normal when put into the medium at the blastula stage, were three which developed into micro-cephalic monsters with a single median eye. The phosphate uptake of these was measured separately and found to exceed to a great extent the uptake of normal embryos (Table 2).

TABLE 2

The uptake of phosphate (counts per minute, c./m.) by microcéphalie embryos of Xenopus laevis compared with that of normal embryos. Mean counts per minute of 32P with standard error; stages after Nieuwkoop & Faber (1956) 

The uptake of phosphate (counts per minute, c./m.) by microcéphalie embryos of Xenopus laevis compared with that of normal embryos. Mean counts per minute of 32P with standard error; stages after Nieuwkoop & Faber (1956)
The uptake of phosphate (counts per minute, c./m.) by microcéphalie embryos of Xenopus laevis compared with that of normal embryos. Mean counts per minute of 32P with standard error; stages after Nieuwkoop & Faber (1956)

Concentration of phosphate, pH, and other conditions

In general the pH of the radioactive medium was adjusted to 7, but in one experiment groups at pH 6 · 0 and 7 · 2 were compared. There was no difference in uptake except that the group at pH 6 · 0 gave more variable results. In the same experiment, media prepared from the same sample of isotope diluted to different concentrations were prepared. Over a range of 3 – 10 μ C. per ml., the ratio of 32P uptake to concentration of 32P in the medium was roughly constant for a given stage, indicating an uptake in direct proportion to concentration of the medium (Table 3). Each value was derived from about ten embryos. A second smaller experiment gave similar results. In the preliminary work with axolotl embryos such proportionality was also found.

TABLE 3

The ratio of uptake of 32P to the concentration of the medium Xenopus embryos

The ratio of uptake of 32P to the concentration of the medium Xenopus embryos
The ratio of uptake of 32P to the concentration of the medium Xenopus embryos

Factors such as the degree of crowding, depth, and volume of the medium, which might affect metabolism and therefore also phosphate penetration, were kept constant for any one experiment, and varied little from one experiment to another. A few experiments in which these conditions were varied showed that phosphate uptake was in fact affected. More crowded embryos, for instance, took up less phosphate.

The effect of injury

When fragments of gastrulae and neurulae were incubated in radioactive media, whole embryos of the same stage were relieved of their vitelline membranes, being punctured in the process, and incubated with the halves for comparison (Tables 6 and 7). In the period from immediately after cutting until 2 hours later, the amount of phosphate taken up was similar to that taken up by the fragments, and was much higher than that which penetrates intact embryos With vitelline membranes. Subsequently the rate of phosphate uptake diminished, though not to such a low level as that of a normal embryo at this stage.

In a few experiments normal embryos in their membranes and injured embryos without membranes were incubated in the same medium and so directly compared. For instance, in one experiment, the uptake of normal embryos, incubated for 2 hours in the medium, was 0 · 9 ± 0 · 2 counts per minute 32P, while that of the injured embryos was 15 · 2 ± 1 · 3 counts per minute (15 embryos in each group, 5 radioactive samples).

It seems unlikely that the vitelline membrane actually hinders entry of phosphate. In the case of axolotl embryos, which can be removed more easily from the membranes, without injury, it was found that taking off the membranes had no effect on phosphate uptake. It cannot be assumed either that the radioactive medium simply flows into the wound, since the first rapid phase of closure of the wound was completed before the embryo was transferred to the medium, and in the experiments with fragments, healing appeared perfect before the third incubation period (i.e. from 4 to 6 hours after cutting) usually even before the beginning of the second incubation (from 2 to 6 hours after cutting). The increased uptake of phosphate appears to be connected either with the exposure at the surface of cells usually covered, or else with the metabolic changes involved in recovery from the wound.

The uptake of phosphate by fragments of Xenopus embryos

The proportion of the original embryo present in each fragment was estimated as follows. Batches of ten fragments or whole embryos were weighed after being dried at 115° C., and the mean dry weight of one embryo or fragment was calculated. The average values for two batches of eggs are given in Table 4.

TABLE 4

Dry weights of half-embryos of Xenopus laevis as per cent, of whole embryos

Dry weights of half-embryos of Xenopus laevis as per cent, of whole embryos
Dry weights of half-embryos of Xenopus laevis as per cent, of whole embryos

The blastulae were cut round the equator, half-way between animal and vegetative poles; the difference in weight is presumably due to the presence of the blastocoel in the animal half. The gastrulae were cut through the middle of the blastopore at the horse-shoe stage, so that the future dorsal halves were separated from the future ventral halves. The neurulae were cut so as to include in the dorsal half all the neural plate except for the tail region, which is curled round ventrally at this stage. Almost all the endoderm was included in the heavier ventral half.

The radioactive medium used for the first experiment, with half blastulae (Table 5) was made up from the same sample of 32P, diluted to the same extent, as that used for the experiment with half-gastrulae, the results of which are shown in Table 6. For this reason these values for the properties of blastulae and gas-trulae are directly comparable. The results for the second experiment in Table 5 (half-blastulae) are for the same reason directly comparable with those of experiment II in Table 7 (half-neurulae). It can be seen that the slight tendency of whole gastrulae and neurulae to take up more phosphate from the medium than blastulae is greatly accentuated when fragments of these stages are used. The halfblastulae take up less phosphate than half-neurulae, and much less than half-gastrulae. These comparative data are shown in Text-figs. 1 and 2.

TABLE 5

The uptake of phosphate by Xenopus half-blastulae expressed in mean counts per minute 32P taken up in the time stated with standard error

The uptake of phosphate by Xenopus half-blastulae expressed in mean counts per minute 32P taken up in the time stated with standard error
The uptake of phosphate by Xenopus half-blastulae expressed in mean counts per minute 32P taken up in the time stated with standard error
TEXT-FIG. 1.

Comparison of the uptake of phosphate by fragments of Xenopus gastrulae and blastulae incubated in the same radioactive medium, A, ventral half of gastrula; B, dorsal half of gastrula; c, vegetative half of blastula; D, animal half of blastula

TEXT-FIG. 1.

Comparison of the uptake of phosphate by fragments of Xenopus gastrulae and blastulae incubated in the same radioactive medium, A, ventral half of gastrula; B, dorsal half of gastrula; c, vegetative half of blastula; D, animal half of blastula

TEXT-FIG. 2.

Comparison of the uptake of phosphate by fragments of Xenopus neurulae and blastulae incubated in the same radioactive medium, A, dorsal half of neurula; B, ventral half of neurula; c, vegetative half of blastula; D, animal half of blastula

TEXT-FIG. 2.

Comparison of the uptake of phosphate by fragments of Xenopus neurulae and blastulae incubated in the same radioactive medium, A, dorsal half of neurula; B, ventral half of neurula; c, vegetative half of blastula; D, animal half of blastula

Two further experiments with half-neurulae fully confirmed the results given in Table 7. One further experiment each with blastula and gastrula fragments confirmed the results of Tables 5 and 6, except for one difference in the blastula results. This experiment was carried out at a higher temperature, so that development was more rapid, and during the period from 4 to 6 hours after cutting the ventral halves formed deep blastopores. During this period the uptake of 32P was not only much higher than that of the animal halves (72 ± 11 c. /m. as compared with 12 ± 4) but also much higher than the uptake during the 0 – 2 hour period (29 ± 5 c./m.).

Half-blastulae (Table 5)

The halves collapsed after cutting, but within 5 minutes had rolled into balls, with a small group of yolky cells protruding at the cut surface. These cells fell away or were covered in within 2 hours, so that healing was complete before the beginning of the second period of incubation (i.e. from 2 to 4 hours after cutting). Sections showed that cell-division continued in the fragments, bringing about the normal diminution of nuclear size, from about 20 μ diameter at the time of cutting, to a value approaching 8 μ, which is the normal nuclear diameter in stages after the beginning of gastrulation, and in adult skin.

Fragments from experiments I and II (Table 5) were kept for several days after cutting. A small abortive blastopore formed in vegetative halves, while epibolic cell-movements in animal halves led to the formation of a frill of tissue in the region of the former equator. In the experiment discussed above, which was carried out at a higher temperature, these signs of gastrulation movements were already visible at the end of the 4 – 6 hour incubation period. This was perhaps related to the increased uptake of phosphate in this last period.

Half-gastrulae

Healing of the half-gastrulae was slower, but cell-division continued normally, and by the end of 6 hours both halves, now completely healed, had an archenteron and a circular blastopore enclosing a small yolk-plug. Ventral halves kept for a further 24 hours showed no further development, but dorsal halves developed into small larvae, deficient only in the abdominal region.

The uptake of phosphate was on the whole similar in the two halves at first, but became relatively much greater in the ventral half during the third 2-hour period (Table 6).

TABLE 6

The uptake of phosphate by Xenopus half-gastrulae, expressed in mean counts per minute 32P taken up in the time stated with standard error

The uptake of phosphate by Xenopus half-gastrulae, expressed in mean counts per minute 32P taken up in the time stated with standard error
The uptake of phosphate by Xenopus half-gastrulae, expressed in mean counts per minute 32P taken up in the time stated with standard error

Half-neurulae

The neural folds of the dorsal halves continued to close, but so much more slowly than in the normal embryo, that after 4 hours closure was in some cases not complete. At the end of 6 hours, however, the nerve-tube was completed, there was a sucker rudiment, and, in the somites, spindle-shaped muscle-cells were differentiating. After a few days these halves formed swimming tadpoles, deformed in belly and tail regions.

The ventral halves remained throughout the experiment as spherical nodules, in some of which sections revealed a short length of axial (tail) structures. Subsequently some of these fragments formed more or less perfect tails, while others remained undifferentiated.

The uptake of phosphate was at first the same in the two kinds of fragments, but fell off much more rapidly in ventral than in dorsal halves (Table 7).

TABLE 7

The uptake of phosphate by Xenopus half-neurulae expressed in. mean counts per minute 32P taken up in the time stated with standard error

The uptake of phosphate by Xenopus half-neurulae expressed in. mean counts per minute 32P taken up in the time stated with standard error
The uptake of phosphate by Xenopus half-neurulae expressed in. mean counts per minute 32P taken up in the time stated with standard error

The effect of p-dinitro phenol on phosphate uptake

It has been shown that p-dinitrophenol (DNP), which is said to uncouple phosphorylation from oxidation, has a strongly inhibiting effect on amphibian embryos at concentrations of 10 – 4 M to 10 – 5 M (Brachet, 1952). Xenopus embryos were found to respond to 10 – 4 M DNP in a manner similar to that of the other Amphibia studied. Development was at first retarded, but even after 4 hours, when the embryos had become stained yellow, recovery was complete a few hours after return to normal culture conditions. After longer periods in DNP recovery was not possible.

Comparison of phosphate uptake by whole embryos in the presence and in the absence of DNP gives somewhat equivocal results, and would appear to depend on the degree of cytolysis. One batch of neurulae showed only a slight difference: controls took up 1 · 8 ± 0 · 2 counts per minute of 32P in 2 hours, DNP-treated embryos 1 · 3 ± 0 · 2 counts per minute (five groups of embryos in each case). In both groups the neural folds closed during the incubation. A group of gastrulae, incubated at the dorsal lip stage, showed a greater effect of the DNP: controls, 8 · 3 ± 1 · 3 counts per minute 32P (six groups), DNP-treated 1 · 5 ± 0 · 2 counts per minute (seven groups). At the end of the 2-hour incubation period the controls had reached the small yolk-plug stage, the DNP-treated from large to medium yolk-plug stage. Another batch of embryos was divided into three groups, one of which was pretreated for 2 hours with 10 – 4 M DNP solution before incubation in the DNP solution which contained 32P. Development in the treated embryos was retarded more drastically than in the previous batch. The results were as follows: controls, 1 · 5 ± 0 · 2 counts per minute 32P taken up; in presence of DNP, 3 · 3 ± 0 · 9 counts per minute; pretreated with DNP, 3 · 0 ± 0 · 5 counts per minute (about fifteen embryos in each group).

After prolonged immersion, when cytolysis was more advanced, the DNP-treated embryos took up much more phosphate than the controls—presumably by adsorption on the dead cells. After 15 hours in 32P medium controls had taken up 12 ± 1 counts per minute (10 separate embryos), those cultured in DNP/32P solution had taken up 393 ± 18 counts per minute (eight separate embryos).

The effect of DNP on phosphate uptake by fragments is much more marked, as shown in Table 8. The DNP did not prevent or retard the healing of the cut surfaces. The DNP-treated blastula-halves formed epibolic frills and groove-like blastopores in the same way as the controls. However, elongation of the dorsal halves of neurulae was prevented by the DNP. These results were confirmed by further experiments.

TABLE 8

The effect of dinitrophenol on the rate of uptake of phosphate by fragments of Xenopus embryos expressed in mean counts per minute 32P per embryo with standard error

The effect of dinitrophenol on the rate of uptake of phosphate by fragments of Xenopus embryos expressed in mean counts per minute 32P per embryo with standard error
The effect of dinitrophenol on the rate of uptake of phosphate by fragments of Xenopus embryos expressed in mean counts per minute 32P per embryo with standard error

In the first place it must be decided whether the entry of phosphate into these embryos can be considered as a metabolic function, or whether it is incidental to a flow of the medium into internal spaces, directly into the archenteron, or through the wound in injured embryos, or indirectly into the blastocoel, e.g. between the cells. Measurements of the swelling of Xenopus embryos during development were made in relation to this point. The diameter of each of about ten embryos was measured at intervals under the microscope with an eyepiece micrometer scale. The volume of the embryo was then calculated. The measurements were repeated on several other batches of eggs, which gave similar results.

It was calculated from these results that the average volume increase from the 32-cell stage to stage 10 (dorsal lip of blastopore) would be 0 · 036 cu. mm. per 120 minutes, that from stage 10 to stage 12 (beginning of neurulation) would be 0 · 045 cu. mm. If the data presented in Table 1 are now considered, it can be seen that in experiments 2 to 5, these volumes of the medium would contain from 48 to 57 counts per minute for the cleavage and blastula stages, and from 60 to 71 counts per minute for the gastrulation stages. These values are considerably in excess of the actual uptake, so that during imbibition of water a part, usually most, of its phosphate content is excluded.

Injured embryos and fragments, on the other hand, take up much more 32P than can be accounted for by influx of the medium, so that this also is evidence of selective behaviour by the cells.

A striking effect of fertilization upon the penetration of phosphate was found by several workers in a number of echinoderm species (Brooks & Chambers, 1948; Lindberg, 1948, 1950; Abelson, 1947; Marshak & Harting, 1948; Chambers et al., 1948; and Whiteley, 1949). Brooks & Chambers (1954) later showed in two species of Strongylocentrotus that there was in fact a time lag of 15 – 20 minutes after fertilization before the 32P uptake began to increase, and the maximum was only reached after 50 – 90 minutes. In the effect of fertilization on phosphate uptake, therefore, the echinoderms resemble the axolotl.

In general it has been shown that after this increase at fertilization the rate of phosphate uptake remains constant in echinoderm embryos (e.g. Villee et al., 1949), with possibly a ‘mitotic rhythm’ in some species (Zeuthen, 1951) though not in all (Whiteley, 1949). As in the case of Amphibia, the isotope was not usually lost once it had been taken up (except by unfertilized eggs, Brooks & Chambers, 1954). Uptake of phosphate by the echinoderm embryos was greatly reduced but not completely prevented by such inhibitors as 4 – 6 dinitro-o-cresol and usnic acid (Abelson, 1947; Marshak & Harting, 1948). These effects were much more striking than the effects of DNP on the penetration of phosphate into the amphibian embryos.

In these respects it can be seen that the obviously functional phosphate intake of echinoderm embryos is more susceptible to changes in outside conditions and metabolic interference than the ‘vestigial’ phosphate intake or exchange of Xenopus embryos.

The phosphate uptake of echinoderm embryos appeared in some species to be correlated with oxygen uptake (Brooks & Chambers, 1954) but this is clearly not so in Amphibia. Brachet (1954) found no increase in oxygen uptake at fertilization (Rana temporaria = fusca) although the R.Q. dropped from 0 · 99 to 0 · 66. The curve obtained by Tuft (1952) for oxygen uptake by Xenopus embryo does not resemble the phosphate uptake curve. Similarly, the results for fragments of embryos do not show any correlation with what is known of differential oxygen uptake in dissected parts of amphibian embryos. For instance, Brachet (1947) and Boell (1948) reviewed experiments showing a similar rate of uptake of oxygen by dorsal and ventral fragments of gastrulae, in contrast to the results for phosphate uptake. Consumption of oxygen was, however, found to be higher at the animal than at the vegetative pole. If there were a correlation between oxygen uptake and phosphate absorption, therefore, the animal halves of blastulae might be expected to take up more phosphate than the vegetative halves towards the end of the 6-hour period studied, when gastrulation movements begin; but this is not so.

It remains possible that differences in phosphate uptake may be related to differences in synthetic activity of the cells. It has been shown in many different ways that synthetic activity becomes important at the beginning of gastrulation (Brachet, 1952) and this is the stage at which most phosphate is taken up, especially by fragments. Kutsky ( 1950) also showed that while the distribution of introduced 32P among phosphate compounds of R. pipiens embryos was unchanged during cleavage, there was at gastrulation a shift of 32P from acid-soluble to acid-insoluble fractions, indicating synthetic activity involving phosphate at this stage. The drop in phosphate uptake at the tail-bud stage, when differential synthesis is proceeding, may be a consequence of the differentiation of the epidermal cells as a more effective barrier, Mezger-Freed (1953) also showed a decrease in phospho-protein phosphatase activity after closure of the neural tube (R. pipiens). There is, however, no immediate correlation with alkaline phosphatase activity, which rises steadily during the development of Xenopus (Krugelis, 1950). The dorsal halves of neurulae may be considered as showing an adaptive increase in phosphate uptake, deprived as they are of their yolk, the main source of larval phosphate.

In fragments of gastrulae, however, it is the dorsal half in which differentiation is most rapidly proceeding, and yet it is the ventral half which, towards the end of the 6 hours, takes up more phosphate. Grant (1954) found no significant differences between distribution of 32P in dorsal and ventral halves of R. pipiens gastrulae labelled as ovarian eggs. Regional differences in respect of phosphate metabolism clearly require further investigation. Experiments are at present being carried out to determine into which organic compounds the introduced 32P is incorporated. Preliminary results suggest that from about 10 to 20 per cent, of the phosphate incorporated into the fragments here studied was in acidinsoluble form, and that almost all of the 32P was contained in the liquid phase of the cytoplasm and in the small granules (microsomes).

The results for phosphate uptake by fragments show some resemblances to uptake of amino-acids as reported by Friedberg & Eakin (1949) and Eakin et al. (1951) especially at the neurula stage, when glycine was much more readily incorporated into dorsal halves than into ventral. Dorsal halves of gastrulae ol R. pipiens and Hyla regilla took up the same amount of glycine as ventral halves, and a little more methionine. Unfortunately the results were not analysed into an initial healing and subsequent differentiating periods, so that differences which were only apparent in the experiments here reported during the 4 – 6 hour period after cutting could not be expected to appear.

Finally, the curious results obtained with microcephalie embryos need further investigation. The great difference in 32P uptake by these abnormal embryos indicates some disturbance in phosphate metabolism. Gustafson & Hasselberg (1951) suggested, as the result of work on the enzyme systems of developing seaurchins, that lithium (which also causes microcephaly in Amphibia) may act as a ‘vegetalising agent’ by an effect on the phosphorylating system. This seems to offer a convenient means of studying the effects of lithium on amphibian embryos, if it should prove that artificially produced microcephalies also take up an excessive quantity of phosphate. Work along these lines is already in progress, and so far indicates that Xenopus embryos do in fact take up more phosphate in the presence of lithium.

In this connexion it is interesting that Brachet (1954), using frogs, found that hybridization and treatment of the sperm with nitrogen mustard altered the rate of phosphate uptake of the resulting embryos as compared with controls. Work of this type may be expected to elucidate eventually the role of the nucleus in the important phosphate exchanges in developing embryos.

It gives me pleasure to acknowledge the help and advice given to me by Dr. Michael Abercrombie during the course of this work.

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