The Effect of Temperature Upon the Diffusion and Autolysis of the Metamorphic and Pigmentation Principles of the Pituitary Gland

The rapid destruction of hormones by autolysis after the death of an animal is a well-known important fact. The destructive effect of death is largely the result of proteolytic enzymes and it varies with different endocrine organs, consequently the purity and activity of the products derived from these organs depends upon the degree to which autolytic and other such destructive agents can be inhibited. This is not a matter of great difficulty when the gland is readily accessible, but in the case of the pituitary, owing to its position, there is an unavoidable interval between the death of the animal and the complete removal of the gland from the skull. During this period the active principles of the gland are exposed to disruptive forces and diffusion from one centre to another may also occur.

Except for a recent account by the author of post-mortem diffusion of the melanophore stimulant between the two lobes of the gland and the effect of exposure upon the activity of anterior lobe extracts, no definite information exists concerning such changes. It is not possible therefore to reach a complete understanding of the many discordant results obtained by administration of different extracts, nor gain an accurate knowledge of the purity and activity of extracts for purposes of standardisation. Further, more details are required of the most suitable conditions and methods for the preparation of extracts which will give uniform results. In order to obtain fresh data bearing on these problems and to supplement existing knowledge, the following investigations upon the influence of temperature on the rate of diffusion and autolysis of the metamorphic and melanophore principles were undertaken.

There is no reason to suppose that, providing the gland itself remains intact after removal from the skull, any reactions that may be in progress within it, will not continue as before, modified only by external influences. Suitable tests made at various intervals after removal would elucidate the course of these reactions as long as the external conditions are known and are uniform throughout. This method was adopted in previous experiments to detect the post-mortem diffusion between the lobes of the pituitary (Spaul 1925), and by repetition at different temperatures a quantitative correlation of the temperature and the rate of diffusion of the principles can be obtained, but it gives no indication as to the effects of autolysis. The actual moment favourable to the beginning of either is unknown, but whereas autolysis would increase with length of exposure, the rate of diffusion would decrease as the distribution in the gland approached a mean value. Hence the more rapid diffusion at the commencement would mask autolysis until such time as the effects of the latter is increased. The quantitative results would therefore represent the difference between the amount diffused and the amount destroyed by autolysis. Such was found to be the case in the work upon post-mortem diffusion, when increasing amounts of the pigmentation principle were found in the anterior lobe up to four hours after extirpation. Subsequently the amount present in the anterior lobe declined; during the whole time, however, a continuous loss occurred in the posterior lobe. The progress of autolysis and diffusion have now been studied by the following technique. The anterior lobe is dissected from the posterior lobe at varying periods after extirpation. From these preparations standard extracts are made, the relative potencies of which are determined and compared with those of entire glands exposed to similar environmental conditions. Evidence of the behaviour of the pigmentary principle may be obtained in this manner, but little information is obtained regarding the autolysis of the metamorphic principle, since the activity of the latter is readily impaired by dilution and by the antagonistic influence of the diffused principles. The two lobes of the glands were therefore separated immediately before exposure and extracts made at the end of equal periods, the conditions of course being identical. No diffusion was possible and hence only the effects of autolysis were observed.

In a preliminary test a series of 10 per cent, extracts were made at 15° C., 25° C., and 35° C., after intervals of 3 J hours, 6 hours, and 20 hours, but although a general impression of the changes occurring was gained, only an incomplete differentiation of the critical phases was obtained. Accordingly, in the actual test 20 per cent, extracts, made at 15° C., 30° C., and 45° C. after 2,4, $612$⁠, and 21 hours, were used, with satisfactory results. Fresh ox pituitaries were used throughout the experiment. Immediately after dissection from the skull, the glands were placed in a refrigerator, a few degrees below zero, as this treatment effectively prevents further change, at least for a time (Spaul 1925). They were then transferred to the laboratory, where the lobes of some of the glands, were quickly separated while still frozen. These were then spread out on clean, white, glazed tiles and placed in an oven kept at constant temperature. At the same time the intact glands were treated in the same way. At the arranged intervals the required number of glands were taken out, some rapidly dissected into anterior and posterior lobes, and extracts made as speedily as possible of (1) the whole glands, and (2) the anterior and posterior lobes of the glands separately. Extracts of the separately exposed anterior and posterior lobes were made at the same time. This operation was repeated with other supplies of fresh, frozen glands under identical conditions, but the temperature of the exposure was altered. In this manner three complete series of extracts were made, (a) exposed at 15°C., (b) exposed at 30° C., (c) exposed at 45 ° C. Each series consisted of the following:

Extracts of the anterior and posterior lobes and the whole gland were made direct from ice at the beginning of the exposure of the other glands. These glands were subjected to no exposure beyond the period between death and being placed on ice after complete removal from the skull; an interval approximately the same for all glands used in these experiments. These extracts would not indicate the progress of any reaction during this interval, but there is every probability that it is about the same in each gland, whatever happens afterwards. In any case nothing can be inferred until exposure has been studied under known conditions, so that the times of exposure were reckoned from this point and these extracts used as a comparative basis. The same method of extraction was used in every case. The weighed glands were minced and pounded in a mortar, transferred to the required amount of warm 0·125 Per cent. acetic acid, boiled for five to ten minutes, filtered at the pump, placed in clean tubes and sterilised by standing in boiling water for an hour before sealing off.

All the series of extracts being complete, the melanophore test was applied to each individual member. Details of this method are hardly necessary here, since full descriptions exist in other publications (Hogben and Winton 1922), (Spaul 1924). Briefly the method consists in diluting the preparation under examination until such concentrations are reached, which are just sufficient, when injected, (1) to darken, and (2) just fail to darken a “bleached “frog. The extracts are diluted with Ringer’s solution and 0-5 c.c. injections given. The weight of the frog being known, the minimal and subminimal doses (and so the threshold dose in c.c.) of each extract were in turn determined for a standard weight (20 gm.) of frog. The results are shown in the following table.

These figures indicate minimum quantities after dilution and hence the actual amount of pigmentation factor present in the undiluted extract is inversely proportional to these values. The smaller the threshold dose the greater the amount of the factor present in the extract and vice versa. By taking the value of posterior lobe extract prepared direct from ice as a standard the remainder can be expressed in terms of this unit.

Before summarising these results it is necessary to consider some of the probable errors in the investigation. In the first place the ratio of the two portions of the gland varies considerably, depending on such factors as age, development, stability of endocrine system, parentage, and general environmental conditions during life. Selection of the animals was not possible and although the number of glands used was great, only four to six were used in each extract; combined with the extreme sensitivity of the melanophore test, an appreciable error is possible even if the difference between the glands is small. A further source of error lies in a loss due to the evaporation which occurs during exposure in the oven. This affects both the weight and the nature of the chemical changes going on in the tissue. The relative degree of evaporation depends on the relation existing between the surface area and the volume of the organ, so that the separated lobes with greater exposed area were especially affected. Again a certain amount of liquid drained from the glands during exposure, particularly from the lobes separated before exposure. The dissection of the glands and the preparation of extracts had to be made as quickly as possible at the end of each period, but exposure made successful separation of the lobes extremely difficult, apart from the risk of the inclusion of superfluous tissue. However, the total effect was not great and did not prevent fairly consistent and reliable results being obtained. Those glands dissected before exposure, as expected, suffered most, since their extracts showed greater deviations and inconsistency, and gave an exaggeration of the influences noted in the others but, nevertheless, with this fault generally in the same direction, they provided useful confirmatory evidence. Many sources of error were beyond control, but where possible every attempt was made to reduce them and bring them within those limits which permit a general inference and even a fairly accurate determination. In any case the attainment of a degree of precision permitting mathematical interpretation is not possible with biological reactions of this type, since their unknown complexities do not lend themselves to the quantitative exactitude.

The main features revealed by the tabulated results can now be summarised:

• (A)

  Anterior lobe (dissected after exposure), (1) There is at first an increase in the amount of melanophore stimulant, which increases with the rise in temperature, but a gradual decrease follows, which is greatest at the highest temperature studied.

• (2)

  The maximum amount of melanophore stimulant present at any time, and the period of exposure before this maximum is reached, decreases with the rise in temperature.

• (B)

  Posterior lobe (dissected after exposure). (1) A rapid initial decrease corresponding to the gain in the anterior series is followed by a slower reduction in the amount of the melanophore stimulant as in the anterior series. These rates increase with temperature.

• (2)

  The amount present at any time is always greater than the amount present in the anterior lobe at the same time, but these quantities approach equality during the first part of the exposure and are nearest at the lowest temperature when there is the longer interval of exposure before the commencement of the succeeding decline found in both series. In the latter period the decrease is slower than in the anterior series.

• (C)

  Whole pituitary. There is a gradual decrease, intermediate between that observed in the anterior and posterior lobe series, in the amount of melanophore stimulant present. This difference increases with rise of temperature.

• (D)

  Anterior and posterior lobes dissected on ice before exposure. There is a more or less uniform decrease with exposure which increases with temperature in each case.

The gain of melanophore stimulant by-the anterior lobe and the loss by the posterior lobe confirms the previous conclusions of post-mortem diffusion and at the same time demonstrates the influence of temperature upon the diffusion itself.

The succeeding decline similar to that found in previous work agrees with that noted throughout the pituitary and in the dissected anterior and posterior lobe series. In these latter series no diffusion is possible, and the loss can only indicate destruction by autolysis. The steeper decline observed in the series from glands exposed after dissection is attributed to loss during preparation. Further, the fact that the decline exists from the beginning of the exposure in these series makes it evident that both diffusion and autolysis start at approximately the same time. In the other series, until diffusion is nearly complete, loss by autolysis cannot be observed although it actually occurs. The earlier appearance of this decline and the increasing rate of destruction in the pituitary and dissected anterior and posterior lobe series illustrate the effect of temperature. The preponderance of the melanophore stimulant in the posterior lobe extracts and its tendency to migrate to the anterior lobe with exposure give further support to the views expressed as to the localisation of this factor in the posterior lobe during life.

The following conclusions regarding the behaviour of the melanophore stimulant can now be drawn:

1. This factor diffuses from the posterior lobe into the anterior lobe, the rate decreasing as the difference between the amounts present in each lobe decrease until the effects of the autolysis become apparent when its destruction proceeds in both lobes. The rate of diffusion increases, but the period during which it is able to mask autolysis decreases, with temperature.

2. Autolysis commences early in the exposure, its effects being slight from four to six hours’ exposure at low temperature, but become more pronounced at higher temperatures (45° C.).

The experiments so far recorded give definite evidence of the effect of temperature upon the diffusion and autolysis of the pigmentation principle and it is now necessary to direct attention to any such effects upon the metamorphic principle. Here a quantitative investigation comparable with the rapid and delicate response of the frog’s melanophores is not possible. So far as metamorphosis is concerned previous work has established the fact that only preparations of the anterior lobe are able to induce this change and they may be either active or inactive (Spaul 1925). There are no means of determining the amount of this principle present in extracts failing to convert an axolotl to the land form and accelerate metamorphosis in Frog tadpoles. Hence the possibility of a reliable graduated quantitative series is very remote.

During this test the axolotls were kept in water in two litre glass vessels at a temperature of 22°-24° C. They were weighed, measured, and fed with raw meat once a week, and the water changed at the same time. Tri-weekly injections of 0.5 c.c. were given. Thus the conditions and methods varied in no way from those usually employed. Only a small supply of large animals was available and in view of the details already obtained by the use of extracts from glands exposed at 15° C. (Spaul 1925), the anterior lobe extracts prepared after four, and six and a half hours’ exposure (before and after dissection) were selected for the test. This series of experiments, though limited, provided additional information of changes in the behaviour of the metamorphic principle.

The results are shown in Table III.

Those failing to change had 24 injections without showing any of the characteristic pre-metamorphic signs and from previous work it was realised that these extracts were not active. The loss of weight is recorded in each case for comparison, but there is no reason to suppose that it is indicative of any activity of the metamorphic principle in those failing to change.

This table shows that, judged by the extension of the period required for metamorphosis, there is a loss in potency with increased period of exposure and with increase in temperature, whether the glands are dissected before or after exposure. At 15° C. the effect appears to be approximately the same in each series but greater at higher temperature in the series of extracts from glands dissected before exposure. The loss of principle, apart from autolysis, involved by the exposure of the dissected glands, has already been commented upon, and it is necessary therefore to explain the loss of potency in the extracts made from glands dissected after exposure at 15° C. which do not suffer such loss in treatment. Here, however, diffusion is possible and a reduction in concentration certain, whilst there is also a possibility of inhibitory action. It is known that the administration of anterior lobe extracts prepared from fresh frozen glands produces complete metamorphosis in an axolotl in about four weeks, although this period depends upon the animal’s size (Spaul 1924). Increased dosage does not shorten the period to any extent, but dilution prolongs it up to a certain point when the minimum effective concentration is reached. Further the presence of sufficient quantities of the posterior lobe, apart from dilution, retards metamorphosis (Spaul 1925).

Again it has been shown in recent experiments that at 15° C. the autacoid is effective up to four hours’ exposure of the gland, but beyond that time the period for complete metamorphosis increases, and after eight hours there is no change. These facts, together with the results recorded here, suggest that the loss of potency of anterior lobe extracts with regard to metamorphosis, as a result of exposure, is due to the reduction in quantity and effective concentration of the metamorphic factor determined by dilution through diffusion and autolysis and the inhibitory influence of diffused posterior lobe principles. Probably the products of autolysis are also effective. Diffusion and autolysis are accelerated by increased temperature and hence the rapid loss of potency observed in these experiments. In this connection it is interesting to contrast the anterior lobe extracts prepared from glands dissected after exposure for four hours at 15° C. with the whole pituitary extract from glands direct from ice. The amount of melanophore stimulant is about the same in each case but only the former is able to transform the axolotl. Evidently the concentration of the metamorphic principle is a deciding factor and further it seems highly probable that principles other than the melanophore stimulant are responsible for any antagonistic effects.

These limitations of the activity of the metamorphic factor contrast with the independence of the pigmentary principle of dilution and inhibition since the presence of the latter can be detected irrespective of the presence of other pituitary principles and other products accumulating during exposure. At the same time they indicate the optimum conditions necessary for the preparation of extracts of the anterior lobe capable of inducing metamorphosis and indicate the probable causes of failure and the divergent results of commercial products. It was concluded from previous work that the preparation of active extracts necessitated the removal of the gland from the skull, the separation of the lobes, and the extraction with dilute acid as quickly as possible after death of the animal. The present record points to the advantages of this method and show the desirability of keeping the temperature as low as possible before the actual extraction is started. No doubt undue prolongation of the interval between the death of the animal and the preparation of the product and the uncertain temperature during this period account for the wide range of variation and lack of consistency in manufactured extracts which is marked, even though various methods and standards of purity are used.

Recently Smith has obtained a retardation of the metamorphosis of the Colorado axolotl by injection of the “anterior hypophyseal fluid,” but using the same commercial extracts as Hogben he confirmed the acceleration obtained by that observer. He suggests therefore that this response is characteristic of the commercial extract only. The present author has obtained metamorphosis with another commercial extract, although he had several failures, and has further described a wide range of melanophore response from them. As a consequence of the experiments described above and the author’s previous work on post-mortem diffusion, it is now possible to put forward an explanation of the opposite effect noted by Smith and point out some at least of the probable sources of error. At the same time conditions and methods favourable to the production of an extract from the fresh anterior lobe invariably bringing about metamorphosis in approximately the same time have been ascertained and the factors and conditions modifying its activity investigated. Hence it now seems justifiable to regard such an extract as giving a characteristic metamorphic response. It is essential that dilute acid be used for the extraction since no response is obtained with extracts in which the medium used is either distilled water, Ringer’s solution, or alcohol. However, posterior lobe principles are extracted by these liquids as they induce a melanophore response, and in suitable quantities retard metamorphosis. The fresh hypophyseal fluid used by Smith darkened the animal and was prepared apparently some hours after slaughter and not in an acid medium so that a retardation rather than acceleration is not unexpected.

The study of the behaviour of these principles exposed under known conditions provides information of the changes occurring in that period previous to the exposure of the glands. Small quantities of melanophore stimulant, always present in extracts of the anterior lobe of the mammalian pituitary, have already been shown to be due to post-mortem diffusion, since this factor is restricted to the posterior lobe in life. This quantity varies considerably and suggests a high rate of diffusion. However the time needed for the expeditious removal of the gland from the skull is by no means constant, and the temperature of the animal is above room temperature for some time after death, so that the variation and rate of diffusion are explicable. De Beer in his account of the mammalian pituitary describes a dense strip of cells in the pars anterior along the cleft, superficially resembling the tissue of the pars intermedia, which, if in existence, would contribute to the amount of melanophore principle in the anterior lobe extracts, but no conclusive evidence on this point is provided by these experiments.

Attention has been confined to the melanophore and metamorphic principles, but other pituitary autacoids exist and there are no grounds for assuming that they do not diffuse or undergo autolytic action in similar manner during exposure at different temperatures, although the rate and necessary conditions may differ. Knowledge of these matters can only be gained by studying the characteristic reaction by which these factors are identified under definite conditions.

This work was done in the Department of Zoology, Birkbeck College, University of London.

In conclusion I wish to express my thanks to Dr Jackson for kindly revising the MS.