612.65.2 ON THE RELATION BETWEEN THE SITE OF INJECTION OF ANDROSTERONE AND THE COMB RESPONSE IN THE FOWL. By A. W. GREENWOOD and J. S. S. BLYTH (Institute of Animal Genetics, University of Edinburgh). (Received for publication 4th September 1935.) I. INTRODUCTION. RECENTLY it has been shown [Greenwood and Blyth, 1935] that cestrone, in its control of plumage pigmentation in the Brown Leghorn castrated male, can not only affect the growing feathers of all regions of the body through the transport of the hormone by the vascular system, but can also be induced to act directly in a localised region through the medium of intradermal or subcutaneous injections of the hormone at a concentration insufficient to cause a general response. The nature of the reaction obtained suggested that the action of the hormone was a direct one upon the feather follicles. Moreover, the distribution of the affected feathers indicated that dissemination of the hormone was by diffusion, the effect gradually decreasing in feathers at increasing distances from the point of injection. Such intensive localised reactions are not restricted to estrone but may be obtained, for example, in the crop glands of young pigeons following the intradermal administration of lactogenic hormone. This method has been employed by Lyons and Page [1 935] who state "Our method of local administration of mammotropin (lactogenic hormone) allows for its detection in a single microgram dose, whereas when given intrapectorally [Riddle et at.] 1 microgram amounts are required." Studies on the quantitative response of the capon's comb to androsterone [Greenwood, Blyth and Callow, 1935] have demonstrated that insignificant differences are obtained when the responses to injections into the pectoral muscles are compared with those obtained from subcutaneous administration in the same region. In these experiments, however, unlike those on the relation of cestrone to plumage coloration, the hormone was injected in a region of the body considerably remote from the organ to be affected. The similarity in the responses in the combs could therefore be related to the efficiency of absorption of the hormone into the general circulation. In the present series of experiments injections of suitable quantities
268 Greenwood and Blyth of androsterone have been made directly into the substance of the comb in order to test whether an increased local response could be obtained. II. MATERIAL AND METHODS. The birds used in these experiments were Brown Leghorn fowls, bred and reared under standard conditions at the Institute of Animal Genetics. (A brief description of the methods of husbandry appears in a previous publication by Greenwood, Blyth and Callow [1935].) With the exception of one bird (D. 62, hatched in 1932) all the capons used were hatched in 1933. They had already been employed for experimentation with androsterone reported in the publication referred to above, and individual birds may be traced by their numbers. The hens and the cockerel used in the experiments are yearling birds, hatched in 1934. The androsterone, which was part of some material made available by Messrs. Ciba, was provided by Dr. A. S. Parkes of the National Institute for Medical Research. The same concentration of hormone (2 mg./ml. in arachis oil) has been used throughout, except in one experiment where a solution of 1y/nml. in aqueous alcohol was employed. In order to make clear the technical procedure followed in injecting the hormone a brief description of the structure of the comb is given (detailed descriptions are available in the publications of Champy and Kritch, 1926, and Hardesty, 1931). In the adult male the great bulk of the comb is composed of two large lateral plates of loose connective tissue in which the interspaces are so swollen and distended by a matrix of mucoid that the whole assumes a certain rigidity. Abundant elastic fibres are also present. Towards the distal parts of the comb these plates are separated only by a narrow zone of dense connective tissue, but towards the centre the space widens and the loose connective tissue immediately above the head is more or less heavily infiltrated with fat. This region, known as the core, also contains the large main bloodvessels to the comb and an abundant nerve supply. Externally the lateral plates are also bounded by a connective tissue zone which is notable for the size and number of capillaries it contains. It is to this complicated network of sinusoid blood-vessels lying immediately under the epidermal layer that the comb owes its bright red colour. The capillaries are connected with the main blood-supply in the core of the comb by small vessels which run directly through the mucoid layer. It would appear probable that the density of the matrix in the latter is responsible for the exertion of a certain amount of pressure on these vessels, and veins being affected by this to a greater extent than arteries, a high pressure of blood results in the surface capillaries, causing their abnormal dilation.
Injection of Androsterone and the Comb Response in the Fowl 269 The effects of castration on the comb are most marked and appear primarily in the lateral plates where the mucoid matrix rapidly disappears and the supporting cells contract and return to an aspect of unmodified connective tissue. As a result of this regression in the mucoid layer, the pressure on the blood-vessels is released, the capillaries contract and the comb pales. The shrinkage of the comb is also accompanied by more rapid cornification of the superficial epithelial cells, giving it a whitish appearance. The comb of the female differs little from that of the male, the same zones are present but their proportions are altered; the mucoid layer is much thinner while that of the fat is larger and extends uniformly through a large part of the blade of the comb. In all birds an attempt was made to penetrate the mucoid plates and deposit the androsterone in the central core of the comb. This was most conveniently done in the castrated birds by inserting the needle of the hypodermic at the base of the comb posteriorly and somewhat to one side of the median line. The injection was made into right and left sides respectively on successive days. With a sufficiently fine needle it was found that leakage did not occur. In the male and female the considerable development of the blade of the comb posteriorly necessitated a slight technical modification. In these, the needle was inserted at the side or anterior end of the comb and the substance was satisfactorily administered if the central core was reached. For injection a tuberculin syringe (1 ml.) graduated in -1 ml. was used. III. COMB INJECTIONS TO CAPONS. A. In this group a preliminary test was carried out on two capons (Table I.). When the comb increases following five daily injections of androsterone were compared with the results from intramuscular TABLE I. Intramuscular injections. Comb injections. No. of bird. 2. Comb increase Comb increase incmm. in mm. in mm. in mm. E. 227 7 9 15 E. 35 1 1 13 (The intramuscular injections were commenced on the 18th of February and the 29th of March respectively, while injections into the comb were begun on the 12th of May. A total of 2 mg. of androsterone was injected over a period of 5 days on each occasion, and the increase in comb size determined on the 6th day.)
27 Greenwood and Blyth injections of hormone of the same magnitude and concentration on the same birds [Greenwood, Blyth and Callow, 1935] it was seen that a greater amount of comb growth had been induced. With the possibility in view that this increased response might be due to the retention of some of the oil solvent in the comb, a further pair of capons were injected similarly with the same dose of sesame oil only for 5 days. Daily measurements failed to reveal any increase in size. B. Comb injections were then undertaken on a group of 5 birds and wnrere controlled by the administration of similar doses of androsterone intramuscularly to another 5-bird group. (Table II. and fig. 1.) TABLE II. Series B. 1. Comnb Injection. -No. of bird. Comb size (mm.). Increase (mm.) in days. 6 /35. 6/6/35. 7/6/35. 8/6/35. 9/6/35. 1/6/35. 1. 2. 3. 4. 5. E. 144 8 83 86 9 95 2 5 8 12 17 156 81 84 87 9 95 97 3 6 9 14 16 2 68 71 79 84 86 3 6 11 16 18 64 76 77 81 84 88 91 1 5 8 12 15 92 8 8 81 84 87 9 1 4 7 1 Average 766-4 812 84-6 88-8 91-8 1-8 4 6 8 12-2 15-2 Daily increase 1.8 2"8 3 4 4-2 3 Series B. 2. Intramuscular Injection. No. of bird. Comb size (mm.). Increase (mm.) in days. 5/6/35. 6/6/35. 7/6/35. 8/6/35. 9/6/35. 1/6/35. 1. 2. 3. 4. 5. E. 165 66 67 67 68 7 7 1 1 2 4 4 34 76 81 83 86 2 4 7 9 12 325 85 86 87 9 92 95 1 2 5 7 1 327 79 79 82 84 86 9 3 5 7 11 346 72 73 73 77 9 81 1 1 5 7 9 Average 75-2 76-2 77-4 8-82- 84-4 1-2-2 4-8 6-8 9 2 Daily increase 1 1-2 2-6 2-2-4 (In both series the total dose was 2 mg. of androsterone (2 mg./ml.) per bird.) While the average comb increase exhibited by the latter group approximated to the expected increase with this dosage, the comb injected group again showed a significantly larger response. Daily measurements of the combs were continued after the cessation of injections to detect any further abnormality of behaviour, but regression appeared in both groups simultaneously and followed the normal course. C. The fact that the injections into the comb elicited a response
Injection of Androsterone and the Comb Response in the Fowl 271 comparable with that obtained when twice the amount of hormone is administered intramuscularly, and that the combs on the average exhibited a rate of growth which must be in the vicinity of their maximum potentiality in this respect [see Greenwood, Blyth and Callow, 1935] led to the determination of the efficiency of smaller doses. Two 2 Zbe bo 5 is~~~~~~~~t 1/m- Days after 1st Injection. FIG. 1.-Comb response of four 5-bird groups. Nos. 1, 3 and 4 received five daily injections of androsterone directly into the comb, while in No. 2 they were made intramuscularly. Total dose in Nos. 1 and 2-2 mg. androsterone in oil, No. 3--5 mg. in oil, and No. 4--1 mg. in aqueous-alcohol. tests on 5-bird groups were made: (1) daily injections into the comb of a dose one quarter the previous magnitude over a 5-day period, and (2) daily comb injections of -2 ml. of an aqueous-alcoholic solution of androsterone containing 1y/ml. for the same period, so that the birds received one-twentieth of the orignial amount. (Table 3 and fig. 1.) The more striking results were obtained from the first of these two tests (Series C. 1) where the amount of comb growth induced equalled almost exactly that resulting from the intramuscular injection of four times the amount of androsterone (Series B. 2). The data resulting
272 Greenwood and Blyth TABLE III. Series C. 1. Comb Injection. No. of bird. E. 24 23 412 536 585 Average (Dose: Comb size (mm.). 1/6/35.112/6/35.113/6/35.1 14/6/35.1 91 73 79-6 95 8 81-97 81 8 8 75 82-6 99 85 82 84 85-6 15/6/35. 16/6/35. 1. 11 13 4 86 89 2 84 85 86 88 8 81 1 87-6 89-2 1-4 Daily increase 1-4 Increase (mm.) in days. 2. 6 3 2 2 2 3 1-6 3. 8 7 4 65 6-3 Each bird received 5 ml. androsterone (2 mg./ml.) daily for 5 days. Arachis oil medium.) 4. 5. 1 12 8 11 6 7 8 1 7 8 7-8 9-6 1-8 1-8 No. of bird. Series C. 2. Comb Injection. Comb size (mm.). 13/6/35. 14/6/35. 15/6/35. 16/6/35. 17/6/35. 18/6/35. Increase (mm.) in days. 1. 2. 3. 4. 5. E. 11 75 77 12 244 Average (Dose: 79 69-8 8 8 69 75 75-6 76 81 81 71 75 76-8 82 82 82 83 71 73 75 77 776-6 Daily increase 1-2 -2 21 1-8 -6 2 4 3 4 2 3 2 2 1 1 2-2-8 1-2 -8 Each bird received -2 ml. androsterone (-1 mg./ml.) daily for 5 days. Aqueous-alcohol medium.) from the second experiment (Series C. 2), while not strictly comparable, since the medium in which the androsterone was administered was different, is of interest in giving some indication of the increased delicacy of the modified technique in the detection of the hormone. IV. 79 7 75 COMB INJECTIONS TO INTACT BIRDS. In view of the fact that previous workers (Champy, 1931, and Juhn, Gustavson and Gallagher, 1932) had reported that they were unable to induce growth in the comb of the adult sexually active male or of the hen during laying periods by the injection of male hormone, the possibility of causing an increase in the size of the head furnishings of such birds by means of the intra-comb technique was next considered. Three females were selected whose record of egg production showed them to be laying well and regularly; of these, two received comb 4 4 44 3 3-8 1-
Injection of Androsterone and the Comb Response in the Fowl 273 injections of androsterone, and the third, intramuscular injections in the pectoral region. The dosage employed was again -2 ml. daily of a concentration of 2 mg./ml. androsterone in arachis oil. In addition, a normal male received daily injections of the same magnitude into the comb. (Table IV.) Instead of the usual 5-day period, the injections were carried on continuously for 15 days; in the male, since no significant change in comb size was shown during the earlier part of the experiment, the dose was doubled from the 11th to the 15th day. TABLE IV. No. of Initial Increase (mm.) in days from 1st injection. b~ird. Sex. Injected. Comb _ size. 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 11. 12. 13. 14. 15. F. 661 C Comb 22 mm. 1 2 1 2 2 2 2 2 F. 264 Comb 138 mm. 1 2 5 6 7 11 14 18 19 18 19 18 19 F. 137 Comb 143 mm. 1 3 6 7 9 12 13 13 13 14 14 16 16 16 F. 698? Intramusc.127 mm. 1 1 1 1 3 3 5 5 5 5 By the 6th day the two females receiving the hormone direct into the comb showed some growth, but this was small when compared with the increase obtained when a group of castrated birds were subjected to a similar treatment (see Series B. 1). Their combs continued to enlarge until about the 9th and 11th day when growth in length and height suddenly ceased. In one bird (F. 264) the cessation appeared to be complete up to the end of the injection period; in the other (F. 137) towards the end of the experiment some slight growth was apparent (fig. 2). About the time when the changes in growth rate occurred swellings became palpable at the base of the comb in the region where the injections had been made. These continued to increase in size until they were readily obvious to casual inspection. In F. 264, whose comb became completely erect and intensely red, the swelling was distinctly circumscribed so that an area 1 to 15 mm. in height along the entire base of the comb stood out three times as thick as the remainder of the blade, though this also was somewhat thicker than in the normal female. In F. 137 the swelling was not so discrete and concerned the greater part of the blade below its line of junction with the comb points, but this may have been related to the fact that injections into the comb were made at times some distance above the head of the bird. The comb did not redden to the same extent as that of F. 264, nor did it become completely erect. Immediately following the cessation of injections both combs began to regress in size.
2 Greenwood and Blyth The female receiving the intramuscular injections (F. 698) showed none of the phenomena described above and over the whole of the injection period it increased in size 5 mm. only, an insignificant amount 2 in combs of this size, which are known to be subject to minor, intermittent fluctuations. to /, "V\ In the male (F. 661), de- + f / ~ ~~\ spite the increased dosage in 1 so "\the /,,2 / latter half of the experiment, no measurable growth in the comb was observed. H,/ During the course of the experiment the birds were weighed three times, but no -*-* '' significant differences were observed in any of them. Days after 1st Injection. That neither the intra- FIG. 2.-Response of two normal females to intra-. comb injections. Dose: -2 ml. of androsterone comb nor intra-muscular in- (2 mg./ml.) daily for 15 days. jections interfered with the Solid line-f. 264. Broken line-f. 137. reproductive activity of the females to any appreciable extent will be seen from Table V. The slight falling off in the number of eggs laid during the period of injection, even if significant, would be as likely due to the daily handling of the birds as to a specific hormone effect. TABLE V. Number of eggs laid (15-day periods). No. of Injection. Pre-injection. Injection. Post-injection. 2/5 to 3/6. 4/6 to 18/6. 19/6 to 3/7. 4/7 to 18/7. 19/7 to 2/8. F. 264 Intra-comb. 13 11 1 12 11 F. 137,, 13 12 11 11 12 F. 698 Intra-musc. 11 13 11 11 1 Total 37 36 32 34 33 It has been found [Greenwood and Blyth, 193] that successful implantation of testicular substance in the intact female may cause, not only an interference with the rate of egg production, but also a modification in the shape of the eggs laid, due to an insufficient production of the oviduct-stimulating hormone by the ovary. The shape of the eggs laid by the females used in this experiment was unmodified by the treatment they received.
Injection of Androsterone and the Comb Response in the Fowl 275 V. COMB INJECTIONS TO A PARTIALLY CASTRATED MALE. The smallness of the responses in the intact birds following a long series of injections with androsterone when compared with those obtained in the completely castrated birds suggested the possibility that the magnitude of the response was inversely related to the initial comb size. To test this assumption an incompletely castrated male, in which the comb had attained a stable level at a size roughly equal to that of a female, was given a five-day series of intra-comb injections with androsterone of the same dosage and concentration as was given to the intact birds. The response was immediate and could be detected 24 hours following the initial injection. By the 6th day the comb showed an increase of 17 mm., that is, equal to the best response elicited in the total castrates treated in this manner. (Table VI.) Following the cessation of injections regression in the size of the comb occurred. TABLE VI. Comb increase Comb size (mm.). (mm incdas. No. of Sex. (mm.) in days. bird. 25/6/35. 26/6/35. 27/6/35. 28/6/35. 29/6/35. 3/6/35. 1. 2. 3. 4. 5. D. 62 152 156 161 166 167 169 4 9 14 15 17 VI. DISCUSSION. That the comb of the Brown Leghorn fowl shows an increased growth response to androsterone when that substance is administered directly into the comb, as opposed to intramuscular injection in the pectoral region, is evident from the results of these experiments. It had been established earlier by Greenwood, Blyth and Callow, on birds from the same stock, that with 5-bird groups a difference of 3 mm. average in comb size could be considered as a statistically significant deviation; in the two 5-bird groups used for comparison of the efficiency of the two injection techniques (Series B 1, B 2) the difference was 6 mm. Confirmatory evidence was also provided by the tests on three intact females, where the visible difference between the effects of the two treatments was greater than the recorded measurements of length plus height of the combs would suggest. That the hormone should prove more effective by this technique (intra-comb injection) is somewhat difficult to comprehend and further experimentation is necessary before the mechanism of its action can be satisfactorily explained. The comparable localised reaction induced in the feathers of the Brown Leghorn castrate by VOL. XXV., NO. 3.-1935. 19
276 Greenwood and Blyth means of intradermal and subcutaneous injections of cestrone [Greenwood and Blyth, 1935] suggested that the characteristic distribution of the effect indicated diffusion of the hormone through the adjacent tissue, the cestrone acting directly on the feather follicles. The similarity between the two reactions is particularly noticeable in the comb injected females; it is true that at first a general increase in comb size was found, but measurement of length plus height affords no evidence of where actual growth took place, whether it was general throughout the whole comb, or whether in fact it was localised in the region where the swellings were later to appear. It is also possible that intramuscular injection results in greater wastage of the hormone, due to excretion of certain amounts from the circulation before it reaches the organ to be affected, or to utilisation of it in conditioning other sex characters. A suggestive fact emerging from these experiments is that in both groups of castrates, whether injected intramuscularly or into the comb, regression occurred at about the same time following the cessation of the treatment. In attempting an interpretation of the phenomena encountered in the intact birds following androsterone administration, two factors must be taken into account: (a) the mucoid plates of the comb are already well developed, and (b) functioning reproductive glands are present. In the first instance it might be argued that in the male the great distention of the mucoid plates inhibited the passage of the injected substance, and that their lesser development in the female produced only partial inhibition, resulting in a localised reaction. Confirmatory evidence on this point was not obtained from the incompletely castrated male where the comb, because of its initial size, must be presumed to have a similar structure to that of the females yet offered no resistance to the passage of the hormone, since the response obtained in this bird was equal to the best elicited in the total castrates. On the other hand, there must be a genetical or mechanical limit to the extent to which combs can develop; while this conceivably might have been attained in the male, earlier experimental work leads to the conclusion that it has not been reached in the female, for in ovariectomised birds, where regeneration of the right gonad has occurred, the comb frequently develops much beyond the size characteristic of the normal hen [Domm, 1927]; similar evidence has been obtained from females with successful testicular implants [Greenwood and Blyth, 193]. With regard to the second supposition, that the gonads themselves may be a factor in determining the efficiency of the androsterone, mammalian studies [Moore and Price, 1932] suggest that the pituitarygonadal relationship is such that while gametogenesis, and the sex characters dependant on the heterologous sex hormone may suffer damage, there is no antagonism to the action of the injected hormone itself. In the present experiments the administration of the male
Injection of Androsterone and the Comb Response in the Fowl 277 hormone did not result in ovarian damage as judged by the number of eggs produced or by their shape (the latter being taken as an index to the state of the oviduct which normally regresses following the limitation of ovarian stimulation). On the other hand, the response to the injection of androsterone was markedly affected by the use of intact animals; the data from the incompletely castrated male does not vitiate the argument here as it did the former one because it is known that castrates with combs of this size may carry relatively small residual fragments of testicular substance. The results obtained indicate a new experience, outside the realm of the theory of Moore and Price; even the earlier hypothesis of Steinach [1926], postulating sex hormone antagonism, would only accommodate the findings in the female and not those in the male. VII. SUMMARY. 1. Injections of androsterone directly into the substance of the capon's comb produced a response greater than that elicited by the same dose when injected intramuscularly in the pectoral region. 2. In a preliminary test of the effect of the hormone in intact birds, two females responding to the treatment, showed less increase in comb size than the capons, as recorded in the length plus height measurements, but developed well-marked swellings at the base of the comb, which are considered to be manifestations of a local effect. A normal male showed no reaction to the same treatment, while an incompletely castrated male, with a comb similar in size to those of the females, gave a marked response. 3. The significance of the results is briefly discussed. REFERENCES. CHAMPY, C. (1931). C.R. Soc. Biol. 18, 367. CHAMPY, C., and KRITCH, N. (1926). Arch. de Morph. 25. DOMM, L. V. (1927). J. Exp. Zool. 48, 31. GREENWOOD, A. W., and BLYTH, J. S. S. (193). Proc. Roy. Soc., B, 16, 189. GREENWOOD, A. W., and BLYTH, J. S. S. (1935). Ibid. 118, 97. GREENWOOD, A. W., BLYTH, J. S. S., and CALLOW, R. K. (1935). Biochem. J. 29, 14. HARDESTY, M. (1931). Amer. J. Anat. 47, 277. JUHN, M., GuSTAVSON, R. G., and GALLAGHER, T. F. (1 932). J. Exp. Zool. 64, 133. LYONS, W. R., and PAGE, E. (1935). Proc. Soc. Exp. Biol. N. Y. 32, 149. MOORE, C. R., and PRICE, D. (1932). Amer. J. Anat. 5, 13. RIDDLE, O., BATES, R. W., and DYKSHORN, S. W. (1932). Proc. Soc. Exp. Biol. N.Y. 29, 1211. STEINACH, E., and KUN, H. (1926). Biol. Gen. 2, 815.