An Experimental Study on the Effect of Thyroxin Upon Sexual Differentiation in the Fowl by A

378

AN EXPERIMENTAL STUDY ON THE EFFECT OF THYROXIN UPON SEXUAL DIFFERENTIATION IN THE FOWL BY A. W. GREENWOOD, PH.D., AND A. C. CHAUDHURI, B.Sc. (From the Animal Breeding Research Department, Edinburgh.)

(Received 14th January 1928.) INTRODUCTION. THE work of Hertwig (1912) on the frog has shown that "over-ripening" of the eggs leads to an effective modification of the sex-ratio in the direction of an increased production of males. Adler (1920) examining the thyroids of the larvae arising from such " over-ripe" fertilised eggs found that they are larger than in normal individuals, and that the differentiation of the thyroid gland occurs at an earlier stage in development than that of the gonad. From this study the hypothesis was formulated that the larger and hyperfunctioning thyroid had influenced the processes of sexual differentiation of the gonad of the genetic female in the male direction. Riddle (1914-1916) and Whitman (1919) working with the pigeon demonstrated that from birds made to produce many eggs during the reproductive season there arise more males during the early part (Spring) and more females later. The male- producing eggs of the early season were found to be smaller than the female- producing eggs elaborated later in the Summer. Riddle regards the male-producing ova of the Spring as ova exhibiting a relatively high metabolic rate and the female- producing ova of the later season as cells of a lower metabolic rate. Riddle and Fisher (1925) in a more recent paper have shown that the period of most abundant male production is the phase of the reproductive season char- acterised by the largest thyroid size in the parent, and these authors conclude that successful modifications of sex-differentiation have been effected through changes in the rate of metabolism in the ova and early embryos. The following experiments were designed with a view to determining whether or not definite modifications were produced in the chicken embryo by injections of thyroxin at a stage of development before the visible differentiation of the reproductive organs has occurred. For the present it is assumed that thyroxin in its action provokes an elevation of the metabolic rate.

TECHNIQUE. Willier (1924) by grafting thyroid tissue on to the chorioallantoic membrane of the developing chick embryo obtained a number of dwarfed specimens. The small size and the emaciated condition of these embryos were regarded as signs of hyperthyroidism resulting in an increased metabolism. In these experiments, however, The Effect of Thyroxin upon Sexual Differentiation in the Fowl 379 it was found that the earliest favourable time for the implantation of the thyroid tissue was the ninth or tenth day of incubation, at a stage, that is, when the sexual differentiation of the gonads had already occurred. To obviate this difficulty the following procedure was adopted in this series of experiments. At the third day of incubation (i.e. at a stage in development when no demonstrable differentiation of the gonads into testis or ovary has occurred) a preparation of the active principle of the thyroid gland (thyroxin) dissolved in saline was injected into the air space of the egg. An alternative method of injecting the thyroxin directly into the albumen was tried but was found to be unsatisfactory, as the increased local pressure produced numerous cracks in the shell radiating from the aperture through which the fluid was injected. The thyroid preparation used was the mono-sodium salt of thyroxin prepared by the British Drug Houses. After removal of the egg to be injected from the incubator, the broad end of the shell was rubbed with a pad soaked in 70 per cent, alcohol, then by means of a fine dental burr a small hole was drilled in the shell in a line at right angles to the long axis of the egg and at a point about 1 cm. from the apex. The solution of thyroxin was then introduced into the air cavity by means of a hypodermic syringe with a fine, slightly curved needle. If the air space had been successfully punctured, the displaced air could be seen bubbling from the hole in the shell. When all the air was displaced the needle was quickly withdrawn, the aperture in the shell sealed with plaster of Paris, and the egg returned to the incubator. The eggs used for controls were subjected to the same experimental treatment save that sterile salt solution was injected instead of the thyroxin solution. Stringent aseptic precautions were taken during the course of the operations. The maximum amount of fluid that can be introduced into the air space of the egg at the third day of incubation was found to be in the neighbourhood of 2 c.c, but there was considerable variation in the size of the air space and usually only about 1 c.c. of fluid was injected; in the table the volume of fluid injected is not stated but the actual quantity of thyroxin injected is given. In the early part of this experimentation, the dose of thyroxin given was calculated from the iodine content of the thyroid of an adult bird per gram of body weight. This was found to be approximately 0-00024 mg. of iodine per gram of body weight. On the basis of the approximate average weight of the eggs used (50 gm.) an amount of thyroxin equivalent to 0-012 mg. of iodine was used. Since the monosodium salt of thyroxin is composed of 61 per cent, by weight of iodine, the dose of thyroxin to be injected on the basis calculated above is approximately 0-02 mg. As an extremely heavy mortality resulted from these early experiments, some reduction in the dose was necessary. Solutions of the following strengths were made up for injection: 1. 0-02 mg. of thyroxin per c.c. of saline. 2. 0-016 mg. „ „ „ 3. o-oi mg. „ „ „ 4. 0*002 mg. „ „ >, BjEB'Viv 25 380 A. W. GREENWOOD and A. C. CHAUDKURI Although these concentrations were productive of a relatively high death-rate in the injected eggs, the dose was not further decreased as a maximum effect on the embryos was desired. The total number of eggs used in these experiments was 226. Of these 190 received injections of thyroxin while 36 received injections of saline only. That the number of controls used was small compared with the number of eggs receiving the thyroxin was due to the fact that the preliminary experimentation showed that the death-rate was likely to be extremely high from these thyroxin injections, and, with only a limited number of eggs available, it was desired to obtain as many experimental embryos as possible. The eggs used for the experiments were from a pen of Light Sussex hens mated with a Rhode Island Red cock (a sex-linked cross) so that the genetic sex of the embryo could be determined by macroscopical examination of the plumage. On removal from the incubator for examination of the embryos at varying periods during incubation, namely the 10th, nth, 16th or 17th days, the eggs were opened and identification of the sex by an examination of the plumage was made. The yolk was then removed and the embryos weighed. This was followed by a dissection and examination of the reproductive system after which the gonads were removed to a fixing fluid (Allen's modification of Bouin) for subsequent histological examination.

RESULTS. It was found that out of a total of 190 eggs injected with thyroxin only 39 embryos survived the effect of the treatment and were alive at the time of examination. That the causal factor of this high mortality was the thyroxin and was not due to the experimental method employed was shown by the fact that the eggs used as controls, which underwent a precisely similar experimental treatment except that saline alone was injected, showed a very much lower death-rate. Of the 36 eggs injected as controls 29 survived until the time of examination. The death-rate in the thyroxin injected eggo was therefore 79-5 per cent, while that in the eggs injected with sterile saline was only 19-5 per cent. No deviation from the normal development of the reproductive system either in the embryos from thyroxin-injected eggs or in the control embryos was en- countered. Further, the sex of the individual as determined by an internal examination of the embryo invariably agreed with the sex as determined by inspection of the down coloration. The weights of the experimental and control embryos obtained are given in Table I, and although the numbers were not very large it will be readily seen that some of the chicks have been markedly affected by the thyroxin. This is not notice- able in those embryos examined in the second week of incubation. The embryos which can be considered as showing symptoms of hyperthyroidism are confined to those examined on the 16th and 17th day of incubation (with possibly one embryo at the nth day of incubation). It would thus seem that the effect of the thyroxin is not reflected in the growth-rate of the embryo until some The Effect of Thyroxin upon Sexual Differentiation in the Fowl 381 point later than the 10th or nth day of incubation. Examination of the embryos which did not survive the injection showed that there were two peaks of mortality, one soon after the injection and the other about the 12th day of incubation.

Table I.

Experimental males Experimental females No. of Controls No. of days in- batch cubated Weight Dose mg. Weight Dose mg. Weight in Weight in in gm. thyroxin in gm. thyroxin gm. (males) gm.(females)

IO 10 2-67 0*016 2-92 0-02 2'6o 2-88 IO 10 294 o-oi 2*77 0-02 2*59 2*29 IO 10 — — 3'2O 0-02 — — IO 10 — — 2-82 O'O2 — — IO 10 — — 3-02 0-016 — — IO 10 — — 2-42 0-016 — — IO 10 — — 3-O5 O'OI — —. IO 10 — — 2-94 O'OI — — 12 11 *2'7O 0-016 — — 3-8 3'4 12 11 4-40 0-016 — — 13 11 4-60 O'OI 4-35 o-oi — 3'5 13 11 3-10 O'OI 4-10 0-005 — 13 11 475 0-005 4-00 0-005 — — 5 16 *I2'5 0-016 *I3'7O O-OO2 16-5 15*35 5 16 18-0 o-oi i6-oo O'OI — 17-00 5 16 2O'O 0-002 15-20 O'OI — 19-20 5 16 I5'8 O-O02 15-70 O-OO2 — — 5 16 *i3'i5 O-OO2 — — 8 16 — *I2'72 o-oi — — 12 17 *i2'75 O'OI — 19*6 22-05 12 17 — — 22*1 — 14 17 18-5 COO25 16-9 O-OO5 19-2 24-50 14 17 *i4'O 0-005 — 20-80 21-90 7 17 *I2'I7 0-02 *io-95 0-016 20-75 21-64 7 17 IP/27 0-015 — — 20-70 17-80 7 17 *8-o7 O'OI — — 21-50 11 17 *9'O2 O'OI — — — 24-35 11 17 •12-15 O-OI5 — — 24-10 2O'2O 9 17 — — 19-8 o-oi 17-65 22'2O * Embryos showing effect of hyperthyroidism marked with an asterisk.

The facts that no marked effect on the injected embryo is shown until at least the nth day of incubation, and also that around this period there is a decided rise in the mortality rate, suggest that the embryo reacts to the thyroxin at a particular developmental stage. On the other hand it is obvious that such results would be also explicable if it could be shown that through physical interference the thyroxin did not become absorbed into the embryonic circulation until this time. As there is apparently no direct correlation between the amount of thyroxin injected and the effect on the embryo, it would seem that an appreciable amount is lost and is not available for absorption into the embryonic circulation. In this connection there are at least three sets of factors to be considered. (1) The earliest stage at which the area vasculosa of the chick has extended sufficiently to bring it into close proximity to that part of the inner shell membrane underlying the air space. 25-2 382 A. W. GREENWOOD and A. C. CHAUDHURI (2) The permeability of the inner shell membrane and layer of albumen to the thyroxin. (3) Evaporation of the fluid from the air space and deposition of thyroxin on the membrane. It therefore becomes necessary to show that, if the favourable conditions for the absorption of the thyroxin depend on the extension of the area vasculosa over the surface of the inner shell membrane under the air space, these conditions are present before the end of the 6th day of incubation when differentiation of the gonads first can be satisfactorily demonstrated. Examination of a number of eggs revealed the fact that at the 3rd day a small crescentic area of the vasculosa overlaps the inner membrane. This gradually increases until at the 6th day the conditions for absorption from the whole surface of the inner shell membrane are realised. Preliminary experiments were made to determine approximately the rate of absorption of fluid through the inner shell membrane of the egg. Absorption was found to take place rapidly. Two and a quarter hours after the injection the air space was found to be nearly dry, while the remaining injected eggs examined after a period of 24 hours were found to have completely absorbed the fluid. Although the conditions of incubation are not such that rapid evaporation of moisture from the egg is conceivable, it was thought profitable to determine approximately the rate of evaporation of fluid from the surface of the egg. For this experiment five eggs were "blown," 2c.c. of saline were injected and the aperture in the shell then sealed. The eggs were placed in the incubator and removed after 24 hours and the volume of the unevaporated saline was determined. The loss by evaporation varied from 30-50 per cent. Since nearly all the fluid in the air sac disappears within 2 hours of injection, the loss due to evaporation need not be considered here. Histological examination of the gonads from both the thyroxin-injected chick embryos and controls was made. There was no indication that the thyroxin affects in any way the normal process of differentiation of an indifferent gonad into ovary or testis. In the female embryo which had received thyroxin, the canaliculisation of the medullary cords, the secondary proliferation of sex-cords from the germinal epithelium, and the formation of the so-called luteal tissue had proceeded exactly as in the gonads from control embryos. Moreover, from an examination of the ovary of control embryos at the 16th or 17th day it was seen that the nuclei of the cells formed by the secondary proliferation had entered into the prophase of the first maturation division. Similarly the nuclei of the secondary sex-cords of the ovaries from thyroxin-injected embryos showed that the same stage of the meiotic prophase had been reached. It is thus seen that far from inducing gross histological modifications in the structure of the ovary, thyroxin in these doses and injected into the air space apparently does not even affect the rate at which the processes of differentiation occur. No histological differences in structure between the testes from thyroxin injected males and control males were found. The Effect of Thyroxin upon Sexual Differentiation in the Fowl 383

DISCUSSION. The results of this experimentation show that it is possible by injecting thyroxin into the air space of the incubating egg to produce dwarfing of chick embryos, this dwarfing being due it is assumed to the raising of the metabolic rate. These results are similar to those of Willier who did not use thyroxin but implanted thyroid tissue from fowls on to the chorioallantoic membrane of the developing chick embryo. It is reasonable at the present stage of this experimentation to accept his conclusions that such modifications as reduction in size and emaciation of the body are to be regarded as hyperthyroid symptoms and mean that metabolism was increased, more particularly an acceleration of catabolism over anabolism, but experimentation relating to the basal metabolism of such thyroxin injected and control eggs is now being undertaken to decide this point. When the bearing of these results on the question of the relation between metabolism and sex-determination is examined, it is seen that neither in the experiments of Willier nor in these of the present series was this increased metabolism productive of any modifications in the processes of sexual differentiation. Assuming that male is to be distinguished from female by a higher metabolic rate, it is of course to be expected on the above hypothesis that this treatment would affect only those embryos which are genetic females producing a modification of the processes of sexual differentiation in the male direction. As the grafts of the thyroid tissue were made by Willier at a time when the differentiation of the gonad had already proceeded some considerable way, it is perhaps not reasonable to expect such modifications to be produced by this technique. As the injections of thyroxin were made at a stage in the development of the embryo at which no demonstrable differentiation of the gonads had occurred, it might be deduced from the results of the present series of experiments on chick embryos that the theory that an increased metabolism tends toward the production of a high sex-ratio has not been upheld. It cannot be said however that the results recorded here can lead as yet to any definite conclusion as to the validity of the hypothesis, for although the injections were made at the 3rd day of incubation no effect on the embryo was observed until, at the earliest, the nth day of incubation. That the effect of the injections does not take place until about this stage in incubation is also shown by the fact that there is a definite peak of mortality in the experimental embryos about the 12th day of incubation. The present paper is of the nature of a preliminary report. It will be necessary to determine by further experimentation whether the apparently delayed action of the thyroxin is a specific characteristic of the substance itself, acting on the developing chick only at a definite stage in development, or whether the results obtained were due to mechanical hindrances by which the absorption of the thyroxin into the embryonic circulation was not effected until some considerable time after the injection of the solution into the air space of the egg. 384 A. W. GREENWOOD and A. C. CHAUDHURI

SUMMARY. 1. An attempt was made to determine in the developing fowl embryo whether injections of thyroxin into the incubating egg would lead to a modification of the processes of sexual differentiation. 2. The injections into the air space were made during the 3rd day of incubation, that is, at a time before the differentiation of the gonads in the two sexes can be demonstrated. 3. Of the 190 eggs injected with thyroxin, 39 embryos survived of which 12 were markedly affected, reduced size of embryos being regarded as an indication of a marked hyperthyroidism which has resulted in an increase in the metabolic rate. 4. No effect on the processes of sexual differentiation in either sex was found. 5. The effect of the injections was not apparent before the 1 ith day. It will be attempted in a further series of experiments to determine whether this is a specific reaction of the thyroxin or whether it is due to mechanical hindrances to the absorption of the thyroxin into the foetal circulation until a considerable time after the injection. 6. These experiments can not at present lead to any definite conclusion as to the relation between increased metabolism and the processes of sexual differentiation.

REFERENCES.

ADLER, L. (1920 a). Pfliiger's Arch. 183, 23. (1920 b). Arch.f. Exper. Path. u. Pharm. 86, 159. HERTWIG, R. (1912). Biol. Centralbl. 32, 1. RIDDLE, O. (1914). Bull. Amer. Acad. Med. 15, 265. RIDDLE, O., and W. S. FISHER (1925). Amer. Journ. Physiol. 72, 464. WHITMAN, C. O. (1919). Publ. Cam. Inst. of Wash. 257, 11. WILLIER, B. H. (1924). Amer. Journ. Anat. 33, no. 1.