Experiments on the Injection of Pituitary Body (Anterior Lobe) Extracts to Axolotls

Abstract

It has been shown that the removal of the pituitary body in mammals is generally fatal. Adler, whose work was extended by Allen, Smith, Atwell, and Hogben, has shown that such fatal results do not attend the removal of the amphibian pituitary body. The former investigators were able to destroy the hypophysial rudiment in the frog embryo, and prove that failure to metamorphose is the inevitable consequence of this operation. This work has been strengthened by the observations of Allen and Swingle on the acceleration of the metamorphosis in frog tadpoles by the implantation of the pituitary gland, and they concluded that the anterior portion (pars anterior) was responsible for this curtailment of the larval period. However, Gudernatsch and others found that pituitary feeding does not stimulate metamorphosis in Anura, and Huxley and Hogben obtained similar results with axolotls after feeding several months with anterior lobe. Later, Hogben found that by injecting commercial preparations of the extract of the anterior lobe axolotls could be induced to transform in a few weeks. Further metamorphosis was induced in thyroidless larvæby the same method, suggesting the direct influence of the pituitary body on the metamorphic changes of the axolotls.For these experiments, undertaken to test the efficacy of anterior lobe extracts as a means of inducing metamorphosis in the axolotl, twelve different products from various manufacturing chemists were obtained, some being in powder, others in tabloid form, and one a liquid extract.* The dried gland was extracted with Ringer’s solution in each case, and each made up to the same concentration, using the manufacturer’s estimate as the basis. The actual strength used was I gr. of dried substance in 5 c.c. of Ringer’s solution, which was equivalent to 5 grs. of fresh gland in 5 c.c. Ringer’s was also added to the liquid extract to make it up to the same strength as the others. Intraperitoneal injections were made tri-weekly, 0.5 c.c. at the time, so that the dose was equivalent to 12 gr. of fresh glandular substance, although in some series it was altered. The axolotls were kept in separate vessels of about 2 litres capacity. Feeding with raw beef, weighing and changing of water was carried out on days alternating with injections. In later stages feeding and change of water took place once a week.The expenses of this research have been defrayed by a grant from the Dixon Fund. The work was carried out at Birkbeck College, University of London, through the kindness of Mr Jackson, who gave facilities and opportunities for the prosecution of these experiments.The first series of experiments consisted in comparing the effects of tri-weekly injections of each extract. The individuals were treated in separate containers, each animal being injected with the same preparation each time, and conditions arranged to give uniformity throughout. Specimens not under treatment were kept in the same environment to act as controls.*List of preparations used, the form in which they were supplied, the quantity of dried gland present and its equivalent of fresh gland, and amount of Ringer’s solution added.The range of temperature for the first part of the experiment was 17° to 19° C., but subsequently it was practically constant at 20° C. Previous to the first injection the weight and length of each animal were recorded, and these observations were repeated twice weekly during the period of the experiment. At the same time fresh water was placed in the container. 0.5 c.c. containing the equivalent of 12 gr. of fresh glandular substance was injected on each occasion, and these injections were continued, except in cases to be referred to later, until twenty injections had been made.There was only one case of metamorphosis in all those animals experimented on in this series of operations (No.7), and in this instance the first signs of change—a reduction of the dorsal fin—was noticed after twenty days at the ninth injection. The reduction of tissue continued until the twentysixth day, when the gills began to get smaller and the skin was cast, giving the animal a lighter appearance. The axolotl itself showed no signs of discomfort, being quite active and feeding normally, although there was a marked loss in weight as shown in table and curve. The length was unaltered. The skin had lost its soft velvety appearance, being quite smooth and of leathery texture, whilst the body itself was more rigid and firmer.At this point owing to exhaustion of supplies, the injections ceased, but after a period of nine days, during which it remained under conditions identical with those of the early part of the work, an injection similar to that given to No. 8 of the series was administered. No further change had been noticed during this break, but soon after this fresh injection the animal became sluggish, much darker, and refused food, in fact it was in the same condition as specimen No. 8, which had died just previously after thirteen injections. Another injection was given, but as there were no signs of improvement the treatment was discontinued. Within three days the animal had regained its previous condition to all appearances, feeding well and weighing exactly the same. Further supplies having now been obtained, injections with the original extract were continued after twenty-one days, and the transformation proceeded, so that in seventeen days the gills disappeared and the change was complete. The skin was shed several times, and two days later the animal was transferred to dry quarters. The results of the treatment with other extracts will be discussed in another part of this work, but meantime a report on a further series of experiments in which No.7 extract was used only will be dealt with. In this series the changes induced, and conditions and other factors, were studied in much greater detail. Six sexually mature axolotls were taken, weighed and measured as before, arranged in three pairs of roughly the same weight, and placed in a small room kept at constant temperature of 24° to 25° C. One member of each pair was injected tri-weekly with 0.5 c.c. a fresh sample of No. 7 extract made up as in previous experiment, the other with 0.5 c.c. of Ringer’s solution to act as a control. Weights and measurements were taken as before and general conditions kept as uniform as possible. After fifteen days the lightest member of the series showed signs of reduction of dorsal fin. This continued, a few sterile eggs being laid meantime, until the twenty-eighth day, when metamorphosis was 1practically complete. However, injections continued as the weight still decreased, but were stopped at twentieth injection, when weight became constant and animal taken from water. The injections in this experiment were continuous without a break, as happened with all members of this series. The control beyond losing a little weight was unchanged. Feeding was normal and the lengths unaltered.In the second pair the first signs of reduction in the pituitary injected member also appeared after fifteen days, but change proceeded more slowly, probably due to greater bulk, and was not so advanced as first pair at the twenty-eighth day. The assumption of adult characters was complete on the forty-second day, just before the twentieth injection. Injections were stopped at this point and the animal taken out of the water. The weight decreased as in previous cases and was not constant until after twentieth injection. The control behaved as before, feeding normally, and there was the characteristic loss of weight. The third pair was the largest, and here the usual initial loss in weight was obtained, but from the fourteenth to thirtieth day it was practically constant.All these changes in weight are shown on the curve. The first signs of tissue reduction appeared on the twenty-second day, and from then on the progress was extremely slow. On the thirty-eighth day the strength of the injection was increased by half. This produced an acceleration, but on increasing the dose on the forty-eighth day by another half, so that actually 0.5 c.c. of the original extract was given, the metamorphosis was considerably hastened and completed on fifty-third day. During the latter period the loss of weight was very rapid. The skin was cast at the twenty-first injection, and actually twenty-three injections had to be made. Here the metamorphosis was very much delayed, and it was only under the stimulus of the larger dose that it could be successfully completed. The control was quite normal and behaved in exactly the same way as the other controls.The preparation used by Hogben was the desiccated product of the Armour Organotherapeutic Laboratory, that which proved efficacious in the experiments here described was supplied by Messrs Oppenheimer as sterile fluid extract. The results obtained are therefore compatible with the view that there is present in the anterior lobe a substance whose physiological activity is such as to promote developmental changes in Urodele larvæ. The question then arises why other preparations employed did not evoke the appropriate response.In all the samples used the amount of gland extract present was given and the equivalent of fresh gland stated. The extracts for injection were made up accordingly, and only this particular extract (No. 7) proved successful in its action, so that in this manner it has been possible to test whether the amount of free extract was actually present in the preparation, and proceeding on these lines any such extract can be examined.When the above series of experiments had been completed, a communication from the manufacturers showed that the extract No.7 was stronger than stated, and on the new calculation 0.75 gr. of fresh gland had been given in each 0.5 c.c. instead of 0.5 gr. However, this does not in any way oppose the of the above for of the active will be later in which a dose of all those extracts which in this series was but with no more that failure was not a question of quantity but is possible to give the of the animals used in these operations were sexually mature and were than those employed by Hogben in The were and these results that of and the injection of quantity of extracts of the anterior lobe will The to gr. 0.5 c.c. injection, from the amount to complete the change of the member of the series of experiments This amount with the as in the same series of experiments, but there is no uniformity in the the amount injected and the Another point from these experiments is the the temperature and the of It is the only in the environment whose could be It was noticed in kept 17° C. that metamorphosis was whilst above the animals became sluggish, food, and losing weight with no in of the temperature is to C., at which the first signs of metamorphosis the to days, and the progress is complete by the day for mature In animals the first were a little and the transformation complete in about but in the the first signs appeared much later, and the dose given was not the for its the transformation was and complete in However, as in the case of the it is to give The temperature and the length of the period of transformation were by an experiment on an axolotl, injected with 0.5 c.c. of No. 7 extract containing 0.75 gr. of gland, kept at temperature of to were in the first change at the day, and a of changes it completed the assumption of adult characters on the possible that increasing the amount of gland in the case of preparations in several after the first series of experiments the same animals to be were injected with twice the same dose of the were similar except the temperature was to 24° C. one two the animals to be the same and there was not one case of this question of metamorphosis a on the of metamorphosis be given as the observations are not in with those of In each case the reduction of the dorsal at the anterior was the first by a of the fin. of the dorsal the became marked and also of the In series the latter was one of the first The reduction of the gills in some cases before the of the dorsal fin. This was generally the case in but they to the being the to In other cases the when the dorsal had the body became considerably and whilst the more rigid and was no in length as measurements will until and then it was very The larval skin was shed early soon after the of the dorsal The of marked the of the changes as usual when the form had been are and no due to greater and but the is the This also to in the of the of these changes and the period which they in body weight of the animals obtained in these experiments were and in to previous In instance there was an initial loss of weight during the first days, the loss being greater in the case of those animals kept at the The continued, but was only until just after the first signs of change when a further marked until the of was The weight then remained more The transformation in the specimen was until the dose was and the in weight was until the later In no instance was there an in weight although feeding was normal and The injected with showed a the experiments, whilst the used in first series of experiments kept at room temperature showed no marked now be to those extracts which to about metamorphosis when and the to for are several an active have been but not in quantity to about the a have been about by the reduction of the of the active during but the experiments in which the of the dose was increased make this very there is the question of having an be due to some other substance present in the pituitary the of lobe one that a time of the from one part of the gland to so that the the the and of the active the greater the of and also the of the two and of the pituitary gland is well and it is quite to that of the lobe as in anterior lobe of the actual experiments carried out with these different commercial extracts will the in anterior extract and the and of the lobe. The results obtained by using No. 7 have been but the of was exactly the same. injections were made with a dose of 0.5 gr. in 0.5 c.c. of Ringer’s were measured and as before and other were the the operations in the first series being at room In all cases the water of the after injection with and 8, and became very and a was The are of each animal No. injections. day, day, day. reduction in tissue at day but no further No in except from to day when after injections injections. constant day when weight increased by to After day and tissue reduction at day. day very marked and this in after to of and of Injections but remained in day when it and and normal except which was in and showed due to of fluid of and some This of fluid for in weight in of of were signs of tissue reduction and were well to activity of anterior lobe but condition of animal further injections. practically same throughout. from to day. No in of injections. and weight injections. and length same. except to injections. loss in weight during experiment. same. injections. in weight after day. day, day, day, No change in after day. No signs of tissue but animal and day, c.c. of fluid for in small no day, animal showed and normal except for was was due to in amount of fluid which to with. were produced in No. 7 as when treated with this injections. and length constant Feeding tissue reduction and injections. in weight of 5 period of experiment same and feeding in weight from to in after day. very in with of and reduction was but animal died after day. showed same as in previous in 12 to of this series were injected with extracts of the being to the amount of anterior lobe extract to about but results were The normal dose was to the and and injections made as in other cases and under the same injections were made. was little in weight except in and which showed and also No. 5 extract was for the except in those particular cases when a desiccated was No. 12 extract. was constant and feeding of these that although no case of metamorphosis there were signs of tissue reduction in there was no in weight except in that died showed due to of no in no of tissue reduction and to about the same time in each case, and no due to same the results of the second series of experiments with No. 7 extract and treated with Ringer’s solution, a third series were at temperature of 24° to 25° with dose of twice of former only were used in this work, for No. 7 had been and 8, had fatal results in the first same animals were used as in the first and during an of the animals had original appearance. had been kept at room temperature and and once a week.The results can be as No. 12 injections. practically length and of water. 12 injections. also feeding normal and 12 injections. reduction in length refused very and 12 injections. reduction in length became of water and 12 injections. in length feeding water and found after injection, having a the fourteenth day all showed signs of tissue The was very but as this change was so it be that it had any with as no further characteristic changes were there was no the injections were stopped on the day. During the operations measured of the water in each were for of but although it was the quantity was small to be successfully experiments, suggesting of free active anterior lobe to about the assumption of adult gave no for in It is possible that the of lobe successfully of the anterior portion in this series of and that with smaller of temperature in the case of No. 7 has been but no was made to the and of the water in the during the experiments at the a condition to a greater in all other cases treated with these extracts at and but not in The of the water at the temperature in some fresh water other day. This appeared to be of the same in each case, and the the of it quite to that the condition was due to the increased activity of a particular which was the same in each case, and its greater activity was by the The fact that the activity of this with different extracts that it be due to a lobe in the anterior lobe the strength of its activity the amount of lobe and further its have the of the anterior injected with the lobe have been to from loss of increased and of the axolotls died as before, and condition previous to in a manner with that described in to In other cases loss of and of was as soon as there was an in the of by and although those cases were not fatal the condition was due to the same This of the and the amount present on these in the injections have been very and the of in the sample of fluid extracted from No. 8, and the of the fatal previous cases to as well as the by all to the amount above observations that an a lobe was present to in 8, and treated with these extracts and 12 have a quantity which could be in the as well as temperature series of experiments, whilst and 7 probably quantity only at It will be that although No. 7 successfully about it some of the but the amount was not to the of the anterior work on the of pituitary extracts on the of a means of the of this in the anterior lobe products used in these The of animals to change and so to has been and of but it is only of that the changes and has been and later induced such changes in with gland to with and Allen obtained of in frog tadpoles by Huxley and Hogben and others have and further obtained in axolotls after pituitary a in with the work of Allen and who obtained after pituitary Swingle the gland tadpoles and obtained a The same was by Hogben and when adult were injected with lobe extract. In a series of a and has been made by Hogben and on the of the The of the to this extract was and with the of in the same and these have been able to the influence of the on the amphibian to environment by of the gland in adult have the same to a of the strength of pituitary extracts by the of the of but the question has now as to the of the extracts made by Hogben from the three of the pituitary gave a so that the of of the strength of lobe the lobe present in anterior lobe on this method, is The of the of more in the and it is quite possible that will for its in other but further is before are the itself was successfully to the amount of present in the original extracts used in this work, and results with a view to its on of the anterior lobe for these experiments were after weighing by and and then in dry with in a dry place on a in In a few to they were quite all the having series of injections were first carried out with a sterile liquid extract containing supplied by Messrs and with to an its Injections were made by various dorsal all the same 0.5 c.c. injections from a series of of were made in pairs of Within there was a which its in about an and the remained for several the dose was but with the were in a environment they regained the condition and were able to to further injections the same so that was complete and they no This with the of Hogben and The was found to on the weight of the so that all results were in of with a weight, uniformity in the The of that solution of which just gave a of the skin was and also the dose which just to give any In this way the and were after to the weight. The animals were the skin being in and in The original extracts were taken, and the and found as in the case of the preparations were also made to with the can at some of the amount of the present in each injection made in the initial series of experiment was carried out with to the of giving by an same was in the case of extracts a to by The experiments were carried out with were injected with 0.5 c.c. of and three gave and no The is the the results of injections made with various of the extracts injected in the experiments, and also the and temperature during these experiments was 20° C. It was found that with the was not and a point was not This have been due to various factors, but in any case they were and only those giving a further was made at the when only those individuals with a skin having a uniform when were taken to The the body weight and dose was found to a but as these were not as but for the small amount of was of no This was therefore and by the of and when referred to the weight, the amount of present in the dose injected during the metamorphosis series of experiments was to The table of the injection of each and the of as by of this table No. 7 extract metamorphosis in but No. extract to the injections having fatal but there is only a of the 8 and have fatal results when but have smaller of this than No and have than No but to about from these that the of a amount of the is of no consequence and does not the activity of the anterior lobe The of the latter can be by the of there is at a amount of free it that the actual which the of the anterior lobe and which has been discussed is not identical with the in the latter have very this actually and its is to from these experiments and further to will be before any can be the so the of this particular a quantity does not the transformation of larval to be to it but in these experiments No. 7 is successful although this and are whilst other extracts when a dose is taken and prove the amount of present being is little of different in the pituitary body. have been but is from and in this case it quite to that these do and that the is not identical with that which responsible for the of the anterior lobe when these results are I to supplies of anterior lobe from manufacturing chemists which successfully to out this lengths and of animals at various stages of treatment with No. 7 at different and lengths and of at same lengths and at same stages of individuals treated with other extracts lengths and at same stages as before of individuals treated with extracts of twice the and at temperature