Comparative Studies of Accelerated Amphibian Metamorphosis

The intimate relationship between metamorphosis in the Amphibia and the endocrine system has demonstrated the significant rôle of the Thyroid and Pituitary, and successive investigations upon this phenomenon continue to emphasise their importance. Extirpation of either or both of these glands from frog tadpoles results in the persistence of the larval phase, whilst a precocious change to the adult follows implantation. Further, the removal of the pituitary checks the development of the thyroid, but hypertrophy of the pituitary has been noted after thyroidectomy. Apart, however, from the evident functional correlation of these glands, the stimulation of metamorphic processes by two members of the endocrine system commands attention and requires closer examination. The administration of thyroid either in solution or by feeding or injection produces accelerated metamorphosis, but only suitable injections of the anterior lobe pituitary are successful. Also the thyroid is able to utilise free inorganic iodine for the same purpose, but the responsible factor in the anterior lobe of the pituitary cannot combine with it in any way to increase its activity in metamorphosis, so that it is apparent these two glands secrete different active principles capable of achieving the same result. This may be the effect of a controlling influence exerted by these glands upon one another, as indicated above in reference to extirpation, but pituitary-less tadpoles are transformed to adults when given thyroid (Allen, Swingle) and injections of the anterior lobe pituitary convert thyroidectomised axolotls (Hogben, 1923). It follows, therefore, that both these active principles can induce metamorphosis directly and independently. This fact makes imperative an examination of the sequence and rate of the changes in metamorphosis induced by each of these factors in different environments with a view to ascertain, if possible, any variations indicative of their different behaviour or activity at any stage before the same final result is produced. By this means an increased knowledge of the characteristics of these principles becomes possible. So far treatment with one or the other has been observed separately, and little if any, comparative work attempted, but, beyond certain qualitative data, the experiments provide limited information, since they are not strictly comparable as conditions and quantities were not the same. To remedy this defect the influence of the thyroid and the anterior pituitary upon metamorphosis of frog tadpoles (R. temporaria) have been studied upon a comparative basis under identical conditions in the series of experiments described here. The same uniform conditions have been maintained throughout any one series of experiments, but the conditions have been altered in each series. Also in view of the relation of iodine to metamorphosis and the accelerating factors of these glands, its influence has been studied in some series and included in the comparison.

The accumulated evidence of many workers has shown that the thyroid can produce accelerated metamorphosis in tadpoles when given either as a diet of fresh gland, dried gland mixed with flour or in powdered form, or as injections of saline, aqueous, or dilute acetic acid extracts, or in solution. The rate of change and the length of the initial period before any acceleration is noticeable depends upon the quantity given, and the size and age of the specimens, other conditions being equal. Tadpoles and axolotls feeding upon fresh anterior lobe pituitary substance increase in size without any tendency to metamorphose but injections of dilute acetic acid extracts are able to bring about this change. Here the rate induced depends within certain limits upon the dose. As regards pituitary treatment in solution, there does not appear to be any definite account, although it would seem that a negative response is to be expected. However, the question has been investigated in the following experiment. 100 c.c. of 0·5 per cent, solutions in tap-water of 20 per cent, extracts of thyroid, anterior pituitary, and posterior lobe pituitary in 0·125 Per cent, acetic acid, were placed in glass dishes together with a dish of tap-water for controls, on a white background at ordinary room temperature (15°−18° C.) with ten tadpoles at approximately the same stage of development (the hind limbs were just visible) in each. The tadpoles had been collected outside and then kept in tanks in the laboratory with a plentiful supply of algae for a few weeks previous to the experiment. The extracts were prepared in the usual way from fresh frozen glands (Spaul, 1925). The solutions were changed daily and no food was given during the period of the experiment. After 10 days those in the thyroid solution had well-developed hind limbs and were reduced in size slightly, whereas no change was noted in the others beyond shrinkage. At 20 days the thyroid tadpoles had practically completed metamorphosis, but the remainder, including the controls, showed no advance beyond a slight increase in length of the hind limbs. As a further test, tadpoles at more advanced stages of development were placed in a 1 per cent, solution of the anterior pituitary extract, but there was no indication of the anterior pituitary as an effective metamorphic agent in solution. Shrinkage was noticed in some cases and the majority died after 10 to 12 days treatment. Another series was.started consisting of 1 c.c. of the following extracts of the anterior lobe extract in 75 c.c. of tap-water: (1) 20 per cent, extract in 0·125 Per cent. acetic acid, (2) 50 per cent, in 0·125 per cent, acetic acid, (3) 20 per cent. Ringer’s extract, (4) 20 per cent, alcoholic (absolute) extract, (5) 20 per cent, distilled water extract. Again no tendency to accelerate was noted after 10 days, although some appeared slightly in advance in the 50 per cent, extract, but it was insufficient to infer any direct action of the pituitary. Higher concentration had fatal effects within a short time. These observations suggest that in contrast to the thyroid this method of administration is unable to create within the animal conditions suitable for precocious change, for even if absorption of the active principle occurs it is insufficient to reach or maintain the threshold concentration for acceleration.

Records obtained so far, whilst showing a relationship between the quantity given and the rate of change, do not permit an actual comparison of the rates induced by equal doses of the thyroid and the anterior pituitary owing to the wide range of experimental conditions and methods. Here similar treatment by injection has been given under conditions maintained constant so far as possible throughout all the experiments to obtain a degree of uniformity providing a basis for comparison. However quantitatively a high standard of accuracy is not obtainable, since there is always an amount of individual variation among tadpoles (see Table I), in spite of uniform conditions, dependent upon various factors such as food, previous environmental influences, the conditions of development, and others which cannot be controlled with any certainty. These difficulties were to some extent overcome and the probable error reduced by using tadpoles from various sources including some reared in the laboratory, and comparing the progress of metamorphosis under the different stimuli in groups from each supply at the same stage of development and in the same environment. After keeping the animals in tanks with a plentiful supply of algae for two or three weeks, batches of ten were placed for the experiment in 100 c.c. of tap-water in small dishes on a white background with plenty of light at room temperature (17° C.). They were given no food during this period, as no satisfactory estimate could be made of the quantity of nutriment taken. Tri-weekly injections of 0·1 c.c. were given and the water in the containers changed at the same time. The extracts of thyroid and anterior pituitary used were of the same strength (20 per cent.), in the same medium (0·125 Per cent. acetic acid), and prepared in the laboratory from fresh frozen glands in exactly the same manner by the method detailed in other papers (Spaul, 1925) for extraction of the latter. In a series of preliminary experiments an attempt was made to determine by this method of injection the age at which tadpoles can first respond to any accelerating stimulus, but the conditions, withholding food supplies, were too rigorous for the earlier stages and no definite information was available as specimens failed to survive the period of the experiment. In all cases, however, a shrinkage was noted which was greater in those under treatment than the controls. Starvation retards development in any case and may prevent further progress, and in these experiments it is evident that the addition of thyroid or anterior pituitary rapidly exhausts any reserves so that after a time no further differentiation is possible as the increased metabolism required cannot be maintained. Work upon other animals has also shown the close relationship between the size, structure and activity of the thyroid and the state of nutrition (Watson, 1912). On the other hand, with adequate food supplies, premature appearance of limb buds can be obtained by treating tadpoles at a very early stage although the chance of completion decreases with younger tadpoles (Spaul, 1924). However, the method of administration required for the anterior pituitary limits the stage at which observations can be successfully made, and with the unknown factors introduced by feeding any such comparative records are of little value. Apart from the illustration of the importance of food and food reserves in normal and precocious metamorphosis, it was found, as a result of this investigation, that the stage with the hind-limb buds just about to appear (stage 1) was the most suitable for beginning a series of observations under these conditions. Accordingly, three series with controls were arranged : (a) thyroid, (b) anterior pituitary, (c) iodine. In (a) and (b) the effect of equal doses (0·1 c.c.) upon the rate of metamorphosis was observed. Each consisted of four groups, in the first of which the 20 per cent, extract was used, but in 2, 3 and 4 the strength was reduced to 10 per cent., 5 per cent, and 2·5 per cent, respectively by dilution with Ringer’s solution. In (c) the influence of iodine in solution, using three concentrations: (1) 1 c.c. of saturated iodine solution diluted with distilled water to 150 c.c., (2) 1 in 300 c.c., (3) 1 in 450 c.c., known to be within the limits in which acceleration and completion of metamorphosis can occur (Spaul, 1924), was studied by placing ten tadpoles in 100 c.c. of each of these solutions. Measurements of the length and breadth of the body and tail were taken periodically. After 6 days those specimens dosed with the highest concentration of the thyroid and anterior pituitary showed approximately the same rate of acceleration and limb development, but whereas the rate decreased considerably with concentration in the pituitary group, so that the lowest (2·5 per cent.) displayed no signs of accelerated change and 5 per cent, very little, all the thyroid treated specimens showed an increase, and the decline with dilution seemed to be very gradual, the difference between each being small. The rate induced by the 10 per cent, thyroid extract appeared to be slightly less than that seen in those receiving the strongest extract in the pituitary series, whilst the 5 per cent, extract gave an acceleration approximating to that with 10 per cent, pituitary extract and the rate with 2.5 per cent, extract was greater than that noted in 5 per cent, pituitary extract. A gradation in the rate of change with concentration of iodine was seen in the iodine series, but the advance in i in 150 was not so great as that produced by the strongest dose of thyroid or anterior pituitary, whilst a slight increase was just perceptible in 1 in 450. Another series consisting of (a) thyroid, and (b) pituitary, with concentrations 20 per cent., 10 per cent, and 5 per cent, for injections (dilutions with Ringer’s as before), and (c) iodine using solutions 1 in 150 c.c. and 1 in 300 c.c., was started in which all specimens had elongated hind-limb buds (stage 2) and later animals having hind limbs showing the main limb divisions (stage 3). Similar results were obtained, but the induced acceleration appeared to increase with age. The animals used in the latter series had not been fed since the beginning of the first series, so that this increase is not necessarily dependent upon the food taken, although a control series, fed until the injections were started, showed a similar but somewhat greater advance in the rate of acceleration with age. It is interesting to note in this connection the period of fasting coincident with metamorphosis, during which time there is a marked decline in the digestive power. The chief points of interest, however, in comparing the thyroid and pituitary groups is the difference between the activity of the two factors, indicated by the different rates, equal reductions in the concentration of the dose produce. The procedure of tri-weekly injections, usual in this kind of work, is based upon knowledge gained by the author of the activity of the metamorphic principle of the anterior lobe. In the first place, to obtain and maintain the maximum rate a given dose can produce, the injections must be given at short intervals and continued till the transformation is complete, otherwise the rate falls off, and the normal rate may be quickly assumed if the dose is small or the period between successive injections too long. Hence the effect of the injections is short and rapid and the principle appears to act as a stimulus which must be suitably repeated to give maximum results. Again, it appears that the acceleration cannot be increased indefinitely with the dose. There is a maximum, and quantities given at each injection in excess of the amount producing this rate are apparently rendered ineffective, since the same number of injections are necessary for the completion of the change, as when the maximum dose only is given. The surplus is probably disposed of by the animal, or, if retained, is metamorphically in an inactive condition, but exactly how this may be accomplished it is not yet possible to state, although it is conceivable, in view of the reduction of the activity of extracts by increasing their pH (Spaul), that the hydrogen-ion concentration may in some way be responsible. Previous determinations have indicated that tri-weekly injections of 0.1 c.c. of a 20 per cent, extract of the anterior pituitary satisfy approximately these maximum conditions for tadpoles of this species. The present records in the pituitary series, with the acceleration approximately in accordance with the quantities given each time confirm this, and the 20 per cent, and 5 per cent, extracts seem to be the upper and lower limits of the effective quantity. The maximum rate of change appears to represent the greatest response the animal is capable of giving to either principle in these 20 per cent, extracts, since the same rates of progress were noted in each. However, with regard to the thyroid, surplus quantities are stored and used as needed until exhausted, or the completion of metamorphosis as shown by feeding experiments demonstrating the quantitative relation of thyroid to metamorphosis (Swingle, Huxley). The rates observed in the thyroid series suggest that considering the small differences between them, the greatest dose (20 per cent, extract) given was more than the quantity required for the maximum acceleration and was not entirely used in the interval between the doses, whilst the second (10 per cent, extract) was just below and possibly exhausted before the next dose. The difference increases with dilution as already detailed, but the absence of proportional agreement between the acceleration and dosage is understandable, for it will be seen from these results that the concentration of thyroid required to initiate the maximum rate of metamorphosis in these animals under the conditions of this experiment, and just sufficient for the interval between the injections, is less than the concentration of the similarly prepared extract of the anterior pituitary. Quantitatively this method, with accumulations and additions and, consequently, alterations in concentration during the progress of the change, is not so successful as the comparisons of the accelerations produced by proportional single doses (or meals) of thyroid, but it does serve to distinguish between the activities and the effects of the two principles. These deductions gain more support from a consideration of the number of doses required and the time before the complete change occurs. In both series the number of injections ranged from three to five (depending on the stage of development at which treatment commenced), extending over a period of six to nine days before completion, using the largest dose and in some cases 10 per cent, thyroid, compared with approximately six to eight injections during 10 to 15 days at the lower accelerations produced by 10 per cent, pituitary, 5 per cent, and sometimes 2.5 per cent, thyroid. More often the earlier stages, given 2.5 per cent, thyroid, required further injections and up to 18 days before reaching the adult stage, but with 5 per cent, pituitary extended treatment was general. The failure of 2.5 per cent, pituitary extract contrasts with the results obtained with the same extract of thyroid and serves to emphasise the difference in the action of these two principles. The strength of the former was below the threshold value of the effective concentration and repetition of the stimulus produced no appreciable change, but the accumulation of the small doses of the thyroid made acceleration possible and completion in most cases before those given 5 per cent, pituitary extracts. Hence an estimate of the lower limit of the effective quantity of thyroid is not possible by this method of continued injection. For similar reasons the times taken for completion were of little comparative value.

In the iodine series 10 to 16 days elapsed before the appearance of adults in the 1 in 150 solution, representing approximately an acceleration equivalent to that induced by the 10 per cent, pituitary and 5 per cent, thyroid extracts, and more than 24 days in 1 in 450. These periods indicate the inability of iodine in solution to obtain the maximum response in tadpoles. In higher concentrations few survive the toxic effects any length of time, but the acceleration produced is still less than the greatest that can be produced by the metamorphic principles of the thyroid and pituitary, which is consistent with previous quantitative work. This result is not surprising, as iodine does not appear to act directly but requires elaboration to some effective organic iodine complex either in the tissues themselves, as shown by the acceleration produced by iodine in thyroidless tadpoles, or through the agency of the thyroid itself, but in any case the amount is small in proportion to the iodine given (Swingle, 1918, 1919, 1923; Spaul, 1925). There is evidence of chemical relationship between this complex and the active factor of the thyroid, which is supported by the close agreement in the changes observed in tadpoles accelerating at the same rate under the influence of either thyroid or iodine (Harrington, 1926).

A high mortality, diminution in size and shrinkage (apart from that imposed by experimental conditions and noted in controls) were features of treatment with the highest concentrations of thyroid. These effects, greatest in those tadpoles first treated at advanced stages of development (stage 3), became less with the reduction in the strength of the dose. They were noted in the higher concentrations of iodine solution approximating to those given the smaller thyroid doses (10 per cent., 5 per cent, and 2.5 per cent.), and to a less extent in those given anterior pituitary. A more adequate impression of the variation in size with dosage and treatment—for the earlier stages at least—is possible by reference to the table containing the altered ratios of the dimensions and the percentage decrease of the dimensions derived from the average of the measurements of the animals of each group taken before the beginning of the experiment and again a few days later after three injections, a period in which there is least reduction in numbers through fatalities or completion of metamorphosis. The dimensions of animals dying within this period were not taken, but the small error introduced thereby does not materially affect the conclusions that may be drawn. Limb measurements are not included in the table, for it was clear that even with uniform conditions there is a decided amount of individual variation in limb development whether metamorphosis is normal or under the same stimulus, making a comparative estimate over this short period unsatisfactory. It will be seen from the table that there does not appear to be proportional gradation with the dose, as the shrinkage of the body and tail absorption is especially marked with the highest concentrations of thyroid. With the smaller doses the tail absorption is not so complete when the same stage of limb development is reached and may be considerably delayed after the appearance of all the limbs. It seems, therefore, that the tail is more susceptible to higher concentrations of thyroid. The hyperthyroid condition of those given the strongest dose has already been discussed, and it is probable that the surplus stored, maintaining a high concentration within the animal, is responsible for the excessive shrinkage and rapid tail absorption. Huxley (1925) obtained similar results and points out that tissue specificity is of equal value in determining a particular reaction with the activity of a particular endocrine organ.

The same lack of proportion in reduction is not characteristic of changes observed in the pituitary series nor is there rapid absorption of the tail. This suggests a varying response of some tissues to these accelerating factors when the same quantity is given or the same rate of change induced, but further data, both experimental and histological, are required before a complete statement is possible.

So far the maximum strength of the extracts used has been 20 per cent, and the corresponding maximum rate of acceleration found, but the effect of higher concentrations has not been ascertained. Hence a further quantitative comparison _Was undertaken. Tadpoles in dishes were arranged as before in six groups under identical conditions and the following preparations in 0.125 Per cent, acetic acid injected, one to each group: (1) 50 per cent, extract—anterior pituitary; (2) 40 per cent, extract—anterior pituitary; (3) 30 per cent, extract—anterior pituitary; (4) 20 per cent, extract—anterior pituitary; (5) 50 per cent, extract—thyroid; (6) 20 per cent, extract—thyroid. The result illustrated the importance of the initial stimulus given by a particular concentration in determining the rate of metamorphic processes. The rate of change in No. 1 was higher than No. 4, which showed the usual rate for this concentration, and the series Nos. 1–4 gradually and regularly graded down, but the difference between Nos. 1 and 4 was not considerable and far from being in proportion to the concentration. The increased concentrations had no toxic or other harmful influence, the few fatalities being equally distributed, whilst there was no unusual reduction in size. The acceleration produced by 50 per cent, thyroid approximated with that observed in No. 1, although a few lagged, but the reduction in size and tail absorption was greater than No. 6 (20 per cent, thyroid), the latter effect being well advanced before the hind limbs were fully developed. As regards behaviour, the pituitary treated animals were quiet and apparently normal, but the quick erratic movements accompanied by rapid tail movements characteristic of the hyperthyroid condition were particularly noticeable in the 50 per cent, thyroid group. Yet another feature of the thyroid group was the peculiar triangular shape assumed by the abdomen with the regression of the gut. This occurred in those given 20 per cent, thyroid, as in the former experiments, about the time of completed metamorphosis, but it was earlier and more marked with the 50 per cent, thyroid. The pituitary series shows no indication of excessive or early absorption of the gut. Evidently, as in the case of the absorption of the tail, the gut is more susceptible to a higher concentration of thyroid and also varies in its response to the factors of these endocrine organs.

This experiment gives further support to the author’s recent comment (1927) upon the significance of the concentration of the metamorphic principle of the anterior pituitary in the transformation of the axolotl to the adult when contrasting the influence of extracts of the anterior lobe pituitary prepared after varying intervals of exposure and whole pituitary extracts.

In previous experiments the response of the tadpole to thyroid and anterior pituitary has been studied in a uniform environment free from disturbing agencies, but normal development and the tissues themselves can be influenced by various agents and processes other than endocrine. It is conceivable, therefore, that accelerated development during or after treatment by such agents might vary and possibly serve to differentiate between the different hormonic effects. The present account deals with the exposure of tadpoles to the action of small quantities of X-ray with subsequent injections of either anterior pituitary or thyroid, whilst others were placed in iodine solution. The animals, collected soon after hatching from the same locality were kept in a large tank containing tap-water and algae. A selection of animals at the same stage of development (hind limbs just visible with a lens) was made later and divided into three groups—the first two for irradiation and the remainder for control purposes. For the exposure the animals were placed in small shallow oblong glass dishes with sufficient tap-water to cover them, but afterwards transferred to larger vessels (with exceptions stated below) and given algae, and once a week small portions of raw meat. The first group of 400 tadpoles was irradiated for about 3 minutes, then batches of 15 were placed in 75 c.c. of each of the following fluids contained in small glass dishes placed on a white background : (1) tap-water, (2) 1 c.c. of saturated iodine solution in 150 c.c. of water, (3) 1 c.c. in 250 c.c., (4) 1 c.c. in 400 c.c. The remainder were removed to the larger vessel. On the succeeding 5th, 7th and 12th days the group was irradiated as. before, the same quantity of radiant energy passing across the specimens as on the first occasion. Only a single batch of 15 was taken and placed in tap-water after the second and third exposures, but the same series of four after the last exposure, together with the remainder in two portions for treatment with (a) thyroid, (b) anterior pituitary. As each batch was taken from the main group a similar batch from the non-irradiated controls and in the same solution was placed alongside, so that finally there were two complete series of exposed and non-exposed animals. On the second day after irradiation tri-weekly injections of 0.1 c.c. were started. For the injection 1 gm. of desiccated thyroid powder (Armour’s) dissolved in 10 c.c. of Ringer’s solution, and a 20 per cent, extract of fresh anterior lobe pituitary in 0.125 Per cent, acetic acid diluted to 10 per cent, with Ringer’s, were used. It will be noted that the same control over feeding and the use of similarly prepared extracts is not continued in this experiment, whilst equivalent doses of each gland are not given. Such conditions are not essential here, for the comparison is between the response of irradiated and non-irradiated specimens to iodine, thyroid and anterior pituitary, irrespective of the quantities given.

The second group of 200 was first exposed immediately after the first group for approximately 10 minutes and again after 7 days for about the same period to the same amount of X-radiation. After each exposure a series of tadpoles in tap-water and the same iodine solutions, were arranged together with controls, but no thyroid or pituitary series were started.

Attention has been confined so far to one stage of development, but the possibility of altered response with age has to be explored. Accordingly, a group of large tadpoles at a more advanced stage (elongated hind-limb buds) were divided into two groups, one for irradiation and the other for control. Two equal exposures, lasting about 10 minutes each with an interval of 7 days between, were given. Series of exposed and unexposed animals in tap-water and iodine solutions were started under the usual conditions after each exposure and also groups prepared for injection with thyroid and pituitary. All groups were under continuous observation until metamorphosis.

The same apparatus, a normal type of gas tube worked with a Ruhmkorff coil, with the spark between the balls about 4 inches, was used for all the exposures. The tadpoles were exposed to the general radiations from this tube, and the distance, kept constant throughout, between the anti-cathode and the specimens was about 24.4 cm. The quantity of X-ray energy passing across the animals was standardised by the following method. An ionisation chamber was placed in the path of a beam at approximately the same distance from the anti-cathode as the specimens, the beam being at right angles to the rays falling upon the specimens and coming from a slit in the side of the lead box containing the X-ray tube. This chamber contained air and was an electroscope as its electrode was connected to a sensitive gold leaf. The rate at which this electroscope discharged was taken as a measure of intensity of the X-radiation falling upon the specimens. The time taken by the leaf to cross sixty divisions of an eyepiece scale were noted and the mean found. The time of exposure divided by this value gave a quantity which was taken as proportional to the total amount of energy in the incident radiation, and endeavours were made to keep this quantity constant during each series of experiments. Hence as the mean value varied, so the actual time of exposure after the first one in each series varied.

Measurements were not taken, but by inspection differences were revealed as the result of the exposure to X-rays. These observations and the conclusions drawn can be summarised as follows :

There was no apparent retardation of normal development in the first group, and except for a slight shrinkage after three (in a few cases) or four exposures, the animals appeared unaltered and developed at the same rate as the controls. There was an apparent earlier development of limb buds in some after one or two exposures, but the variation was too small and inconsistent to be significant.

In the second group shrinkage was very definite after the second and last exposure, otherwise there was no change in external appearances compared with controls and the rate of development was the same.

The large tadpoles (3rd group) showed decided shrinkage particularly after two exposures, and although many appeared to show retarded limb growth it was slight, and in view of individual variations and other possible factors causing fluctuations there was not sufficient justification for suggesting at this stage the responsibility of irradiation. Later, these specimens recovered and others seemed to accelerate temporarily. Further exposure would probably have provided more definite evidence, but since the major object was to detect any change in the reaction to the thyroid and pituitary hormones after treatment these observations were not extended.

In other respects all the animals appeared to be normal. However, the shrinkage observed, when compared with the unexposed controls, points to some influence of X-radiation, which increases with exposure, and tadpoles in the more advanced metamorphic stages show greater susceptibility.

The progress of metamorphosis, the diminution in size and mortality, depended on the iodine concentration, and judged by the close agreement stage by stage between the exposed and unexposed animals in the first group, the previous exposure to that particular quantity of X-ray does not appreciably disturb the response to iodine. There was the characteristic sequence of changes, and specimens in 1 in 150 were complete in about 21 days. However, a higher mortality and additional shrinkage, most marked in the higher concentrations, was noted in the exposed series of the second group. In the third group these differences were still more marked, whilst a distinct retardation of metamorphic change was apparent. When transferred to iodine after exposure all specimens were approximately the same stage as the controls, but then their rate of development became slower, for the majority of the controls acquired fore limbs first and metamorphosed 4 to 5 days earlier. In the 1 in 150 the mortality was so high that only two survived to complete metamorphosis. Here there is evidence of the influence of the exposure to X-rays in the altered response to iodine administration.

A greatly accelerated rate of change was obtained in all specimens under treatment, but although a strong dose was given, inducing a rapid change, the irradiated series were slower than the controls. In the groups of small animals (1 and 2) all controls with some exceptions in the first group had fore limbs and completed the change before those exposed. Almost all the controls of the large specimens (3rd group) were complete in 6 to 7 days after three injections, but 8 to 9 days were required by those exposed having the same number of injections, and others having four injections took 9 to 11 days. The marked reduction in size, shrinkage and high mortality noted were greater in the exposed than unexposed series. It is evident from these details that irradiation alters the susceptibility of tadpoles to the thyroid stimulation.

A rapid assumption of adult characters was obtained here with the control series faster than the irradiated, but the acceleration was less than that produced in the thyroid series, as the quantity given per dose was less. The “lagging” of the exposed animals was more noticeable in this series. Whereas five injections in 10 to 12 days were sufficient for controls in the large series, seven injections in 15 to 16 days were necessary for those exposed to X-rays. The hind limb development was slower in the latter group and the fore limbs appeared after those of the controls. There was not a high rate of mortality, but more deaths occurred in the exposed groups. There was scarcely any diminution in size or shrinkage in either series beyond that already recorded in the exposed group, the result of irradiation. The retardation observed in the first group was slight, but increased in the second group. Hence after treatment with X-rays specimens are unable to respond as readily to anterior pituitary extract.

These observations upon the reduced ability of tadpoles to respond to agents accelerating metamorphosis after irradiation suggest that it has a depressant action which lowers the capacity of the organism or its tissue to respond to stimulation. It depends upon exposure and has a greater effect at the more advanced stages of development. As to the specific nature of this influence, and whether it effects the metabolism directly or through its action upon the thyroid or other regulating mechanism of the body, it is impossible to state until extended study under varying conditions and dosage, together with histological examination, has been carried out. It would seem from the close agreement obtained in a macroscope examination, and comparison stage by stage of the sequence of the metamorphic processes induced in both exposed and unexposed animals, that all parts were affected to more or less the same extent, but until information correlating the quantity of irradiation, stage of development, and dosage of hormone is available the possibility of selective action cannot be dismissed. Incidentally, this approximation in the sequence of metamorphosis, coupled with details of mortality and shrinkage, give no definite evidence of varying response after exposure to either thyroid, pituitary or iodine, and afford no further means of distinguishing between the active principles. The retarded limb growth after exposure observed in those undergoing normal development is probably further evidence of the depressant effect, and apparently with this dosage the effect is temporary. With smaller dosage and younger animals any effect is smaller and recovery quicker. Recovery in those stimulated cannot be detected with certainty owing to the rate of acceleration, but a small number of specimens in the first group after the four exposures were given injections at intervals of four days. Metamorphosis, as expected, proceeded at a slower rate, but it was about the same in the similarly treated controls and also there was a slight increase in size. This case suggests a degree of recovery from the effects of irradiation, possibly under the stimulus of the injection, but further data are required.

McCord and Marinus (1918), experimenting with tadpoles of the Bull Frog (Rana catesbiana), observed that weak irradiations are without demonstrable effect upon normal tadpoles. They distinguish a slight but distinct increase in the susceptibility of young tadpoles to thyroid stimulation by feeding. They suggest large doses would produce an opposite effect and retard metamorphosis, since, according to Richard’s theory, a small dose of the rays serves to increase the activity of certain enzymes, while large doses produce the reverse effect. This effect would be marked in older animals. However, whilst it is interesting to note, in view of this statement, the retardation recorded here with accelerating hormones and the negligible effects on normal tadpoles, the evidence is insufficient to subscribe to the views put forward explaining those changes.

Recently Huxley (1925), investigating the effects of thyroid and iodine upon tadpoles in suitable concentrations of potassium cyanide and alcohol, observed the inhibition of all metamorphic processes and a differential effect upon limb growth compared with the body and tail. These results prompted a study of the influence of the anterior pituitary upon tadpoles in conjunction with these agents, and, to facilitate comparisons and so the detection of differences in the activity of the metamorphic principles, a similar series given thyroid was included in the experiment. Ten specimens were placed in glass-covered dishes containing 75 c.c. of various concentrations of KCN in distilled water and others in different percentages of absolute alcohol in distilled water in the usual constant environment at room temperature. No food was given and the solutions were changed daily. Tri-weekly injections of 0.1 c.c. were given, using fresh gland extracts (20 per cent, in 0.125 per cent, acetic acid) of thyroid and anterior pituitary. In a preliminary experiment to determine the lethal dose of KCN and alcohol these extracts were found to be too strong, so they were diluted with Ringer’s to 10 per cent. The possibility of altered response with age was considered and three groups of animals used, each group being at different stages of development, (a) without hind-limb buds, (b) limb buds just visible, (c) elongated limb buds, but all members of the same group were approximately at the same stage.

Lethal dose was estimated at N/75,000, in which few survived 24 hours. Three series, for animals at the three stages of development, were arranged, (a) control, (b) anterior pituitary, (c) thyroid, containing the following solutions: N/100,000; N/125,000; N/175,000: N/200,000. There were also controls in distilled water. The observations extended over 14 days and the results in each series can be summarised as follows :

These animals, compared with those in distilled water, showed retarded development and limb growth, and reduction in size. They were most pronounced in the advanced stage at the highest concentration, but decreased with age and concentration except for an apparent slight acceleration in some of the youngest specimens in the most dilute solution. In many cases a stage was eventually reached at which no further progress appeared to be made.

Accelerated metamorphosis, compared with controls in distilled water, was retarded. The difference was scarcely apparent at the lowest concentration but increased with age and the strength of the solution. There was a reduction in size in the higher concentrations and several deaths.

As in the former series retardation graded with the concentration was found. The acceleration, compared with the pituitary, was greater, but the retardation more marked. There was a greater reduction in size and a very high mortality.

The lethal dose was approximately 1.5 per cent. Three series were started as before, with controls in distilled water, of the following solutions: 1 per cent., 0.5 per cent, and 0.25 per cent.

Inhibition of growth and development, and shrinkage were noted, depending on the concentration, but all stages seemed to suffer to the same extent.

The inhibition of precocious changes, diminution in size and shrinkage depended on the concentration and seemingly was the same at all stages. The mortality was very high.

Here again the acceleration was inhibited in proportion to concentration. Diminution in size and shrinkage was marked. Few completed metamorphosis.

These records show that both KCN and alcohol in suitable non-toxic concentrations exert a similar inhibitory influence upon accelerate changes induced by the anterior pituitary to that found in the case of thyroid (also Huxley, 1925). In all cases the effect is dependent upon the concentration, but only with KCN is it possible to detect altered susceptibility with age, the high mortality and excessive shrinkage in the alcohol series preventing conclusive observations. Normal development is similarly affected. It is evident that this influence is due to the depressant effect of these agents upon metabolic processes and in this respect it appears to resemble the effects due to irradiation. No measurements were taken, but in both thyroid and pituitary series a decided retardation in limb growth and delayed tail absorption was observed in the higher concentrations of both KCN and alcohol similar to that recorded by Huxley (1925). These experiments provide no further data regarding the activity of the metamorphic factors of the thyroid and pituitary serving to distinguish them. After the experiments the animals were transferred to tap-water and fed on algae when they recovered and proceeded to develop normally.

The absorption of thyroid from solution by tadpoles under narcosis with subsequent signs of precocious change has already been demonstrated by Huxley (1925). He concluded that the numerous changes involved—increased growth rate and differentiation and retrogression—occur independently of the higher centres of the central nervous system, any effect that they may exert being secondary. It would be interesting, therefore, to discover whether there is a similar independence of the accelerated metamorphosis produced by the anterior pituitary in animals under narcosis. Urethane was used as the anaesthetic. In the experiments the dilutions, 1 gm. in 250 c.c. of distilled water, 1 in 300, and 1 in 350 were used, all of which produced immobility in the specimens for some days, but whereas those in 1/250 failed to respond when touched, a slight movement could be obtained in 1/300, which was increased in 1/350. Further dilution shortened the period of narcosis. There were no deaths except a few in 1/250 after 5 or 6 days, but the mortality increased in higher concentrations. The animals were collected outside and kept in a tank with algae till needed. In each experiment there were three series, (a) control, (b) anterior pituitary, (c) thyroid. There were also tap-water controls. Tri-weekly injections (0.1 c.c.) of the usual laboratory acid extracts of anterior pituitary and thyroid were given, the first injections being made the day after the tadpoles were placed in the narcotic solution. Uniform conditions were maintained at room temperature and no food given during the experiment. Ten specimens were placed in 75 c.c. of each solution in covered glass dishes. In the first experiments all three concentrations were used and tadpoles with hind limbs just visible were selected. Signs of accelerated change were noted in both thyroid and pituitary series in 3 days in all concentrations, but there were several deaths in the thyroid series. After 4 days all undergoing thyroid treatment had died, but at this stage there was definite evidence of the progress of metamorphosis in both series at a rate corresponding to that observed in the tap-water controls. Next day all those given anterior pituitary in 1/250 were dead, but another 4 or 5 days passed before the remainder died. Their rate of change became less after 5 or 6 days. A similar decline was noted in the narcotised controls compared with those in tap-water after progressing together for about 10 days. It seems that prolonged narcosis has some detrimental influence. In the second experiment larger tadpoles having elongated hind-limb buds were selected and only two dilutions, 1/300 and 1/350, used. Here again acceleration was obtained, and there was little to choose between the normal and narcotised specimens, but after 8 days the latter were behind. The mortality was higher in the thyroid, those in 1/300 surviving 5 days and those in 1/35° 11 days, the last survivor in this dilution being almost complete. Those given anterior pituitary in 1/300 survived 6 days and there were four (nearly complete) in 1/350 after 11 days. The controls behaved as in the previous experiment, hence there is apparent normal differentiation in metamorphosis for the first few days of narcosis, but completion is delayed in all cases and few survive to reach the most advanced stages when these changes are stimulated by thyroid or anterior pituitary treatment. The effect depends on the strength of solution and the age of tadpoles. The similar acceleration produced with thyroid in normal and narcotised specimens confirms Huxley’s results, whilst those obtained with anterior pituitary suggest that its influence upon metamorphosis can likewise occur independently of the central nervous system. A curious feature of the development of the narcotised animals in this experiment was the slenderness of the limbs which grew out and remained fully extended.

This comparative study of accelerated metamorphosis has added to our knowledge of the characteristics of the hormones of the thyroid and anterior pituitary, but in this concluding experiment the occurrence of these factors in the lower Chordates is considered. The thyroid is present in all Vertebrates, and its evolutionary histology shows that it has developed originally from the endostyle organ found in the more lowly Chordates—Tunicata and Amphioxus. In view of the known influence of the thyroid and the ease with which it can be demonstrated, it is of interest to enquire into the capacity of this endostyle to show similar activity. For this purpose the endostyle, pharynx, and mantle of fresh Ascidians (Ciona intestinalis) were dissected out, washed and weighed. The same quantity of each (0.75 gm.) was suspended in separate beakers of tap-water, placed on a white background, and kept at room temperature. Twenty-five tadpoles at the same stage of development (hind-limb buds just visible) were next placed in the beakers. A control of 25 tadpoles was given the same quantity of dogfish thyroid. The tissues remained suspended overnight, when they were removed, the water changed and algae put into the beakers. After 8 days all those fed with dogfish thyroid had well-developed hind limbs, but scarcely any advance was observed in the others. Later the former completed their change, whilst the remainder showed no tendency to accelerate. In a second experiment under similar conditions larger quantities of the Ascidian tissues and a second meal after 6 days were given, but there were no signs of acceleration after 14 days. It seems, therefore, that the endostyle, although homologous with the thyroid, secretes no principle identified by its effect upon metamorphosis, or its equivalent, which is undoubtedly correlated with the difference of function. Histological studies of the course of development of the thyroid show a direct metamorphosis of the endostylar organ with great modifications of the original endoderm cells and atrophy of some before the formation of the characteristic vesicles (Marine, 1922).

The subneural gland of these animals is regarded by some morphologists as the homologue of the Vertebrate pituitary, but while the ingrowth from the buccal cavity may represent the hypophysis, the gland itself can only be the pars nervosa of the pituitary (De Beer, 1926). Hogben and Winton (1922) prepared extracts of the gland but failed to obtain any indication of the presence of the melanophore stimulant. Other effects characteristic of the Craniate pituitary have not been obtained by extracts, but this failure neither confirms nor denies necessarily the homology of the subneural gland with the pituitary. The case of the endostyle and the thyroid indicates the value of such evidence in this question.

A survey of these experiments in which attempts have been made to examine the activities of the metamorphic principles under standardised conditions favourable to qualitative and quantitative comparisons, makes further distinctions possible between them.

The utilisation of iodine in solution by the axolotl, when thyroid but not when anterior pituitary injections were given, was the first indication of a constitutional difference. It has been established that inorganic iodine itself does not function as a hormone in metamorphosis, but its power to combine through the thyroid or some other agency, or both, with some organic complex within the body to give some substance closely similar to the active principle of the thyroid, makes acceleration possible. The agreement in the rate and changes observed here in iodine and thyroid induced metamorphosis compared with the pituitary effects, leaves little doubt as to the close relationship of the responsible factors, apart from the evidence from other sources. The acceleration observed in thyroidectomised tadpoles in iodine solution shows that a high concentration is necessary to produce sufficient quantities for metamorphosis, but, as might be expected, lower concentrations produce the same rate in normal animals, since their own thyroid is functioning. In axolotls the threshold concentration of the factor does not appear to be reached in non-toxic concentrations of iodine unless thyroid is also given, although Huxley (1925) has obtained pre-metamorphic symptoms in some cases. [The metamorphosis of the axolotl is however possible apparently in some cases with susceptible strains under suitable conditions by the implantation of iodine crystals in the abdomen (Hirschler, 1919; Blacher and Belkin, 1927), in which case the effect would be equivalent to the transformation of tadpoles in iodine solution.] This fact explains the results obtained when anterior pituitary is given, for there is no co-operation between iodine and the anterior pituitary equivalent to combination of thyroid and iodine. Tadpoles given anterior pituitary in iodine solutions accelerate under the influence of two separate factors acting independently, but in the similarly treated axolotl only one such factor (anterior pituitary) is effective, the other, if present, being insufficient to exert any influence.

Metamorphosis produced by certain commercial preparations of the anterior pituitary (E. R. and M. M. Hoskins, 1920) has been found to be due to contamination with iodine (Smith and Cheney, 1921), but this fact does not invalidate the metamorphic influence of fresh gland extracts. The activity of commercial extracts and factors tending to reduce their potency have been investigated (Spaul, 1924,1925), and in consequence suitable methods for the preparation of active extracts of anterior lobe pituitary from fresh glands devised. Their effects, as the present record shows, is distinct from that of iodine. Also no trace of iodine has been found in fresh bovine and sheep pituitaries (Simpson and Hunter, 1910).

In experiments carried out so far it has been found that only acid extracts of the anterior pituitary show any capacity to accelerate metamorphosis, and injection is the only satisfactory means of producing a sufficient concentration of hormone within the animal, compared with the thyroid which can be administered equally well by mouth, injection or in solution with pronounced results, yet quantitative determinations on these lines have yielded positive evidence of difference in behaviour in the production of metamorphosis as well as showing the greater activity of the thyroid. The concentration of the dose decides the initial rate of change, but there is a limit to the effective quantity given per dose, whilst the time for completion depends on the total quantity of thyroid given, and the number of doses, quantity given per dose, and the interval between, in pituitary treatment. In the one case storage of thyroid occurs and the effect is cumulative, whilst a repeated stimulus, which must be within certain quantitative limits, is required in the other, and if the factor is stored it appears to be inactive. The mortality increases more rapidly with thyroid as dosage increases and the reduction in size is greater. Abdominal shrinkage is another feature of higher thyroid dosage, an outward indication of the greater susceptibility of the gut to the higher concentration. This differential response of tissues seen in the regression of the gut and also tail absorption to concentration of thyroid is another distinguishing feature as a similar lack of uniformity is not characteristic of pituitary treatment. Definite data for a study of the response of the limbs to these factors could not be obtained satisfactorily owing to the individual variation, but it should be noted in this connection that other workers have observed equal retardation in limb growth following extirpation of the thyroid or the buccal ingrowth of the hypophysis or both (Allen, 1919, 1925).

No further means of distinguishing these principles was revealed in those experiments made in conjunction with other agents. Small doses of X-rays had no appreciable influence upon normal development owing to recovery, but accelerated change was inhibited. Alcohol and potassium cyanide reduced the rate of change, the latter also displaying differential effects. Hence whereas the thyroid, iodine, and the anterior pituitary accelerate metamorphic changes, these agents by their depressant action retard these same processes. Under narcosis acceleration by either thyroid and pituitary proceeds normally, so that in producing their effect both are independent of the central nervous system, but their relation to iodine, the quantitative and qualitative differences in their action, and the differential tissue response in the one case give ample evidence of distinct physiological activity.

It has been suggested that one gland, the anterior hypophysis, is concerned with the constructive phase of development and growth, whilst the thyroid is responsible for changes characterised as catabolic. The metabolic influence of thyroid is manifest, but before any adequate definition of the character of the changes directly associated with the activity of either gland is attempted or the extent to which the activity of the one may be dependent upon or determined by the other is ascertained, further studies, including comparative histology, are needed.

These results raise the question of the functional substitution of the pituitary for the thyroid. The experimental evidence of metamorphosis under the stimulus of added quantities of one gland by injection or transplantation after the extirpation of the other, point to independent action, but the failure to change normally after the removal of one or both together with histological studies indicate some kind of interrelation and interdependence which is an apparent necessity in metamorphosis of normal individuals. Also the activity of these glands within the animal is not confined to metamorphosis alone, and although they may be able to act, when present in sufficient concentration, independently in this change, otherwise they may be dependent, but at present knowledge does not warrant an exact statement upon the vicarious relationship of these glands.

These experiments were carried out in the Zoological Laboratory, Birkbeck College, University of London, and I wish to acknowledge commercial supplies of anterior lobe (Oppenheimer) and thyroid (Armour). Further, I am indebted to Dr Jackson for kindly revising manuscript and Mr Dobb for photographic work. The irradiations were performed by Dr Temple, to whom grateful acknowledgement is made, and also to Professor Griffiths of Birkbeck College, for his courtesy in providing facilities for this part of the work in the Physics Laboratory.