THE TESTIS AND THYROID IN A HEN-FEATHERED SILVER-GREY DORKING COCK

73 THE TESTIS AND THYROID IN A HEN-FEATHERED SILVER-GREY DORKING COCK BY GWYNNETH BUCHANAN, Zoology Department, University of Melbourne. (With Plates V and VI.) EARLY in the year 1925 Prof. W. E. Agar obtained a hen-feathered silver-grey Dorking cock, of which he has supplied me with the following details as to its appearance and history*: This fowl was formerly in the possession of Messrs H. A. Bartlett and Sons, Pimpinio, Victoria, who stated that as a cockerel it exhibited no peculiarities, except that when used in the breeding pen in its first season it failed to fertilise any eggs at the beginning of the season, though later on in the same season it is believed to have done so. When it was two years old it assumed hen-feathering, the change being practically complete after a single moult. It was at this stage that the bird came into our hands. The sexes in the silver-grey Dorkings differ from one another as regards plumage colour in the following principal features: Neck feathers: S, (hackle) white;?, grey with narrow silver border. Back and saddle: cj, white; $, finely stippled light and dark grey. Breast: cj, black; <j>, salmon red. Wings, Upper coverts (wing bow): <J, white; $,' finely stippled grey. Lower coverts (wing bar): tj, black; $, finely stippled grey. Quill feathers: S, black and white; $, dark grey. In all these points the hen-feathered cock became practically identical with the female, with the exception that there remained a good deal of white in the wing quill feathers. In shape, the feathers of the hackle, saddle and tail are larger than in the female, but much nearer the female than the male type. The comb had been dubbed before the bird came into our hands. The wattle, however, remained male in type. In behaviour the bird was distinctly male. Confronted with another cock he immediately attacked it. He was assiduous in calling hens to share food. Occasionally he performed some of the movements which frequently precede copulation, such as spreading the wing down and circling, but no actual copulation was ever observed. Eighteen eggs, laid at intervals during a period of six weeks while he was with the hens, were incubated, but all proved sterile. Hens showed by their behaviour to him that they recognised him as a male. The bird was killed for examination after it had been in our possession four months. Since the secondary sexual characters are recognised as being influenced by the internal'secretions, it was thought that an examination of the endocrinous organs ^phis bird might prove interesting. It was accordingly killed on June 23rd, We are indebted to Dr A. W. Greenwood, of the Animal Breeding Research Department of* the University of Edinburgh, for directing our attention to the existence of this animal.

74 GWYNNETH BUCHANAN being at the time in apparently normal health. For purposes of reference jjjis bird will be denoted by the letter H. ^^ On dissection the body cavity was seen to be gorged with fat developed in the omentum and against the dorsal body wall, where it was in such quantities as to displace the renal and reproductive ducts to the middle line, and render them difficult at first to demonstrate. The gonads were paired, in the position of the normal testes, small, oval and red (compared with the usual creamy yellow), and the surface covered with irregular, yellowish, follicular excrescences. The adrenals and pancreas were apparently normal. The thymus- was well developed, but showed no abnormal appearance. The thyroid was in the normal position at the base of the jugular vein, conspicuous, red and hard, with irregular excrescences on the surface as compared with the smooth, soft and rather pale organ of the normal controls. The pituitary body, on examination after hardening, showed no apparent peculiarity. METHODS. The testes, thymus, adrenal, thyroid and pituitary body were removed. The last was fixed while in the skull in Miiller's fluid; the other organs in Bouin, Flemming and Zenker. Sections were cut at 15/x, 10/x and 5/A and stained with Mallory's and Mann's connective tissue stains (Boring and Pearl (o) and with iron haematoxylin and eosin. The pituitary body was stained with Delafield. It was found that Zenker was the most satisfactory all-round fixative, and iron haematoxylin and eosin, and Mann the most useful of the stains. The material fixed with Flemming did not colour easily with connective tissue stains. For control purposes, two normal cocks were.killed. From one (ivj) the endocrinous organs were removed and treated similarly to the above, from the second (iv 2 ) only the gonad and thyroid were taken. HISTOLOGY. Nothing unusual was observed in the microscopic structure of the thymus, adrenal or pituitary body of the bird H as compared with these structures in the normal cock. The testis (Figs. 1 and 2), however, as was expected from its macroscopic appearance, was most abnormal. Spermatogenesis was only observed in certain of the tubules, and then of a very degenerate character. In many cases the lining cells were reduced to a single columnar layer in which pigment was occasionally developed; in others the tubules were more or less full of loose masses of cells apparently sloughed off from the walls in the process of disintegration. The round follicular patches visible on the outside were found to consist of encapsuled masses of cells frequently displaced by pat^ft of strongly fibrosed tissue (Fig. 1). The interstitial tissue was very abundant and the whole structure highly vascular the blood spaces being particularly well-marked

Testis and Thyroid in Hen-feathered Silver-grey Dorking Cock 75 in the albuginea. Masses of "luted" cells were present, their arrangement and fi^bent proximity to the more normal tubules, together with the presence of a basement membrane (Nonidez), showing their evident derivation from the sexual cords. On the other hand, the large and often fibrosed encapsuled masses appeared to consist of interstitial tissue (which, according to Morgan, gives rise to luteal cells). The thyroid (Figs. 3-7) from its macroscopic appearance was also expected to show abnormal histological structure. From sections it was seen that practically the entire organ was affected. The whole gland was very vascular (as was also the case in the normal control cocks). In some sections normal vesicles were present, the lining cells being very much flattened with indistinct inner edges; but many showed marked hyperplasia. Professor P. MacCallum, who was good enough to examine the slides, considered that this appearance pointed to an attempt on the part of the functioning cells at compensatory action in order to make good the effects of the pathological conditions obtaining in the greater portion of the gland. These conditions he divided into two main groups: (1) cystic at one end, and (2) myxoedematous at the other, while irregular, apparently ossifying, strands were found running through a large area of the organ. The cystic part of the gland (Figs. 3 and 5) showed large haemorrhagic spaces caused by bleeding into the vesicles which here tend to coalesce. In this portion the contents of the coalesced vesicles frequently included epithelial cells. The blood masses showed all stages of disintegration, in some cases red cells, and in others phagocytes being present. Blood corpuscles also appeared in the vesicles of the normal cock, though in very small amounts. The cells lining the vesicles were markedly swollen and hyperplastic. The myxoedematous portion of the gland (Figs. 6 and 7) was highly vascular and contained much fibrous intervesicular tissue in which were embedded many small atrophic vesicles. The apparently osseous strands (Figs. 3 and 5) showed little histological detail beyond a deeply staining matrix containing abundance of fibres running for the most part longitudinally, and small cells lying in lacunae, resembling those of connective tissue. The appearance of such a tissue in the substance of the gland may be regarded as pointing to a general condition of hypo-activity. CONDITIONS IN THE SEBRIGHT BANTAM. The condition of the thyroid in the hen-feathered cock H led to a consideration of that in the Sebright bantam, in which the cocks are normally hen-feathered. Accordingly, two Sebright cocks (S t and S 2 ) were killed and the thyroid, thymus and testes of both and the adrenal of one were fixed in Zenker. For purposes of control six normal brown bantam cocks {B x,b 2,B 3,B t,b 5, B 6 ) were also killed and their thyroids fixed in the same way. ^Both birds S t and S 2 were sexually-mature, functioning cocks as shown by the germination of eggs fertilised by them, though the percentage of fertility was low, as is common with Sebrights. In each case, particularly in S lt the body was fat

76 GWYNNETH BUCHANAN in the omentum, between the muscles and in the dorsal wall, but not to an excessive amount in either bird. The testes in both cases were normal in position and appearance, and on sectioning showed active spermatogenesis, the tubules being large and rather widely open, and the intertubular tissue only normally developed. In no sections was "luteal" tissue observed. The thyroid in 5 X was normal in position, at the base of the jugular vein, small, poorly vascular, pale and distinguished from the thymus with difficulty. In S 2 it was also rather small, closely surrounded by fat and by pieces of thymus. The blood supply in this case was good. In the controls, B 2 was also very fat in the omentum and body wall. The remainder showed no excessive deposits of fat. In general, the thyroid, which was rather poorly vascular, was slightly larger on the left than on the right. HISTOLOGY. In S t the thyroid (Figs. 8 and u) was encapsuled in a fairly thick but not very close fibrous sheath. The vesicles were normal in appearance though variable in size, but averaged smaller than in the controls and contained much colloid and, in some cases, blood corpuscles. A small mass of vesicles close to a large blood space showed a rather hyperplastic appearance, but the substance of the gland was very poorly vascular. There was also rather more intervesicular tissue than in the controls (a condition ascribed by Swale Vincent (i 8) to varying physiological conditions). Altogether, the appearance was one of a healthy but very small and poorly vascular gland, i.e. the amount of thyroid activity would be small. In S 2 the thyroid (Fig. 9) was also surrounded by a definite capsule, less developed than in S ± but much more marked than in the controls, and, from this, fibrous trabeculae grew into the substance of the gland, which was very compact. There were many large, irregular blood spaces towards the centre, and the whole structure was more vascular than in S t. It also contained more intervesicular tissue. The vesicles themselves varied in size, but were, as in S lt on an average distinctly smaller than in the controls, and their walls much thicker than either S ± or the controls, almost approaching a hyperplastic condition. Again, the appearance of the gland suggests a healthy but under-functioning structure. In the controls (Figs. 10 and 12) the thyroid was surrounded by a definite but thin capsule, not nearly as well developed as in either Sebright. The gland was very vascular with a good intervesicular blood supply and little intervesicular tissue. The size of the vesicles varied, but was on an average distinctly larger than in the Sebrights. Their walls were thin and their contents occasionally included blood. There was in some cases a marked tendency for the vesicles to coalesce.

Testis and Thyroid in Hen-feathered Silver-grey Dorking Cock 77 DISCUSSION. Crew (3) describes a testis of similar external appearance to that of H in a Buff Orpington hen which became cocky; and Nonidez (13) shows that ligation of the vas deferens or other obstructing conditions will cause extensive degeneration of the testis tubules, reduction of the wall to practically only the Sertoli cells, and liquefaction of the spermatogonia. He also shows (14) that blood stasis, abnormal tubules and pigmentation are common in the testes of hen-feathered cocks. In the case cited by Crew, however, both male and female reproductive ducts were found, whereas in H only the male were present. On the other hand, the large fibrosed masses apparently developed from the interstitial tissue might, by their mere pressure, have caused the degeneration found in the rest of the testis of H by producing a condition somewhat comparable to that described by Nonidez. Morgan's original hypothesis do that the "luteal" cells of the Sebrigh't testes are responsible for the henny plumage of the cock has been criticised by Goodale (7) and by Pease (is), the latter pointing out that the'' luteal" cells are present in varying amounts in the testes of all male birds at certain times; while Greenwood(10) has shown that they have no relation to hen-feathering. None were found in Sebright S t or S 2 in the present case. The facts (1) that castration of hen-feathered cocks causes them to assume male plumage (Morgan (u) and Punnett(i6)); and (2) that castrated birds of both sexes assume male plumage indicates that the hen plumage, whether of the normal female or of the hen-feathered male is correlated with the presence of a secretion of the gonad (of the ovary in the normal female and of the testes in the hen-feathered male) though, as has been pointed out, it now seems that the "luteal" tissue is not responsible for this secretion. Goodale and Nonidez (9) remark that in nearly all adult hen-feathered males the amount of intertubular tissue in the testes is large compared with cock-feathered males, and suggest that this has some relation to hen-feathering. Firket(s), however, considers any secretion produced by the interstitial tissue is not enough by itself to determine secondary sexual characters. The amount of intertubular tissue in the testes of H was excessive, but even if it were sufficient to have caused the henny plumage, a cause for its development must be sought. Nonidez (12) suggests that hen-feathering in roosters is most likely due to the dys-functioning of some endocrine gland, probably concerned with alterations in metabolism, e.g. the thyroid or pituitary. The abnormal condition found in the thyroid of H points to an under-functioning gland which would be accompanied by profound disturbances of the general metabolism of the body. The excessive deposits of fat indicate such a disturbance; deposits of fat are characteristic of feminised male birds (Goodale (8)). The Sebrights S x and S 2, both with small and apparently under-functioning though otherwise healthy thyroids, each had much fat laid down. In the case of H, Si and S 2 the condition of the thyroid was one of either pathological or "healthy'»

78 GWYNNETH BUCHANAN under-functioning, as evidenced by the myxoedematous and cystic appearance on the one hand, and by the small size of the gland, poor vascularity, thick 1 small vesicles, and rather hyperplastic cells in the others. The evidence of the present case therefore suggests that the thyroid must influence plumage in conjunction with the gonad, either by altering its secretions directly (as suggested by the abnormal testes of the hen-feathered Dorking) or by acting in combination with the gonad secretion. The facts, as far as known, can be described symbolically by supposing a quantitative factor determining sex; and a scheme of the physiological determination of the secondary sexual plumage {plumage only) character of fowls can be constructed, analogous in its quantitative character with the schemes proposed by Goldschmidt(6) for Lymantria or by Bridges(2) and by Schrader and Sturtevantd7) for Drosophtla, except that the influence of the thyroid has to be taken into account in fowls. On such an hypothesis the gonad and thyroid exert an antagonistic action, deficiency of gonad influence leading to maleness, deficiency of thyroid influence leading to femaleness. We can arbitrarily represent the relative potency of these influences as follows: Normal testes + 3,, ovary + 6 thyroid - 5 (minus because it is antagonistic to gonad) Degenerate thyroid - 2 (Sebright, hen-feathered Dorking cock). The character of the plumage depends upon the algebraic sum of the influence of the gonad and thyroid. If the sum is + 1 or over, the plumage is female. 2 or under, male. This hypothesis (for which I am indebted to Prof. Agar) assumes nothing in regard to the nature of the influence of the gonad and thyroid except that they are antagonistic. The various possibilities on this hypothesis are tabulated as follows: Castrated <J or $ Normal cj 9 Sebright <J Castrated Sebright S Dorking <$ with degenerate thyroid Influence of gonad 0 + 3 + 6 + 3 0 + 3 Influence of thyroid -5-5 -s - 2-2 -2 Total result -5-2 + 1 + 1-2 + 1 Nature of plumage Super <J & 99 <J or super cj 9 The term super male in the above table appears to be justified, since there seems to be general agreement that the castrated fowl, whether male or female, exhibits the ordinary male plumage characters in an exaggerated form e.g. hackle, saddle and tail sickles are even longer than in the normal male. Finlayu) has recently performed a series of experiments in which ^ testes were grafted into birds of the opposite sex, the original gonads of the host being removed in some cases, and allowed to remain in others. Thus he had four

Testis and Thyroid in Hen-feathered Silver-grey Dorking Cock 79 of birds castrated males with implanted ovaries, males with ovaries in ^^n to their own testes, and the two corresponding classes of female birds. The results were complicated by regeneration of extirpated gonads, and also by a tendency of grafted ovaries to develop testicular tissue, but in general we can say that the plumage develops the sexual characteristics of the dominant gonad. At first sight it might appear that some of his birds must be taken as evidence against the scheme suggested here. For instance, No. 8, a female, had two testes grafted subcutaneously. The plumage of this bird remained mainly female, with slight male tendencies, although dissection showed that it contained a normal ovary and two testis grafts containing motile sperms. It might appear then that this bird should have been a "super female," as it was producing both ovarian (+ 6) and testicular (+ 3) secretions. It is possible, however, as Finlay himself suggests, that the presence of ovaries and testes in the one animal may interfere with the secretion of both. Torrey and Horning (19) have obtained henny plumage by feeding thyroid to normal male fowls. Unfortunately only a short abstract of their paper is available for reference, but it is evident they regard the thyroid as interacting with the gonad to produce the result, but in the opposite direction to the view put forward above. The experiment of feeding thyroid to Sebrights, which we hope to undertake in this laboratory, should throw light on this question. My thanks are due to Prof. P. MacCallum, of the Pathological Department of this University, and to Prof. W. E. Agar for suggestions, constructive criticism and advice, and to whom I am indebted for the hypothesis of the "quantitative factor" controlling plumage. SUMMARY. A case is described of an apparently normal silver Dorking cock becoming henfeathered. The endocrine glands of this bird were examined, and the testes and thyroid found to be distinctly abnormal. The testes showed large masses of "luteal" cells, encapsuled fibrosed masses, abnormal spermatogenesis and non-functioning tubules. The thyroid showed myxoedematous and cystic conditions with an apparent attempt at compensation in the functioning tubules, and on the whole the appearance of an under-functioning gland. The glands of two fertile Sebright bantams were examined. The testes were normal, without "luteal" cells; the thyroid was small, relatively rich in connective and poor in glandular tissue, with small secreting vesicles and poor blood supply, suggesting that the Sebright thyroid is subnormal in activity. The evidence suggests that an under-functioning thyroid, either pathologically induced or apparently healthy, may be a controlling factor in hen-feathering, either bucting in combination with the gonad secretion, or by altering it. ^Prhe hypothesis is put forward that the thyroid and gonad act antagonistically in this respect; and a suggestion is made by which the condition of the plumage may be represented as the algebraic sum of the influence of the two glands.

80 GWYNNETH BUCHANAN REFERENCES. (1) BORING, A. M. and PEARL, R. (1917). "Sex Studies, IX." Anat. Rec. 13. (2) BRIDGES CALVIN B. (1921). "Triploid Intersexes in Drosophila melanogaster." Science, N.S. 54. (3) CREW, F. A. E. (1923). "Sex Reversal in the Fowl." Proc. Roy. Soc. B, 95. (4) FINLAY, G. F. (1925). "Studies on Sex Differentiation in Fowls." Brit. Journ. Exp. Biol. 2. (5) FIRKET, J. (1920). "Recherches sur 1'organogenese des glandes sexuelles chez les oiseaux," Part 1. Arch, de Biol. T. 30. (6) GOLDSCHMIDT, R. (1923). The Mechanism and Physiology of Sex Determination. (7) GOODALE, H. D. (1918). " Intertubular Tissue in the Testes of certain Birds." Am. Nat. 58. (8) (1918). "Feminised Male Birds." Genetics, 3. (9) GOODALE, H. D. and NONIDEZ.J. F. (1924). "Luteal Cells and Hen-Feathering." Am. Nat. 58. (10) GREENWOOD, A. W. (1925). "Gonad Grafts in the Fowl." Brit. Journ. Exp. Biol. 2. (11) MORGAN, T. H. (1919). "Genetic and Operative Evidence relating to Secondary Sexual Characters." Carnegie Inst. 285. (12) NONIDEZ, J. F. (1924). "The Intertubular Tissue of the Testes in Normal and Hen-feathered Cocks." Am. Journ. Anat. 34. (13) (1924). "The Effect of Ligation of the Vas Deferens on the Structure of the Testes." Am. Journ. Anat. 34. (14) (1923). "The Origin of the so-called Luteal Cells in the Testes." Am. Journ. Anat. 31. (15) PEASE, M. S. (1921). "Note on Prof. T. H. Morgan's Theory of Hen-feathering in Cocks." Proc. Camb. Phil. Soc. 21. (16) PUNNETT, R. C. and BAILEY, P. G. (1921). "Genetic Studies in Poultry. Ill, Hen-feathered Cocks." Journ. Genetics, 2. (17) SCHRADER, F. and STURTEVANT, A. H. (1923). "A Note on the Theory of Sex Determination." Am. Nat. 57. (18) SWALE VINCENT (1924). Internal Secretions and the Ductless Glands. (19) TORREY, H. B. and HORNING, B. (1922). Proc. Soc. Exp. Biol. and Med. 19. DESCRIPTION OF PLATES V AND VI. a.v. atrophic vesicles; b. blood corpuscles; b.v. blood vessels; b.c. blood capillaries; c. colloid; cap. capsule; d.t. degenerating tubules;/, fibrous intervesicular tissue;/.6. fibrosed mass; h.s. wall of haemorrhagic space; h.v. hyperplastic vesicle; i.t. intertubular tissue; /." luteal" tissue; n.v. normal vesicle; o. bony strand; sp. tubules in which a form'of spermatogenesis is going on. All outlines are drawn with a camera lucida. Figs. 1 and 2 are of the testis and Figs. 3 7 of the thyroid of the hen-feathered cock H. Figs. 8 and 11 are of the thyroid of the Sebright S t ; Fig. 9 is of the thyroid of S 2 ; and Figs. 10 and 12 of the thyroid of a control bantam B-,,. PLATE V. FIG. I. Showing capsulated fibrosed masses, "luteal" masses and testes tubules in stage of degeneration (Leitz obj. 9, oc. 2, Tube 170). FIG. 2. Showing details of Fig. 1 (Leitz obj. 6, oc. 2, Tube 170). FIG. 3. Showing cystic portion with a large haemorrhagic space, hyperplastic vesicles and two bony masses. To one side are a few apparently normal vesicles which seem to merge into the capsule of the cystic portion (Leitz obj. 3, oc. 2). FIG. 4. Showing normal vesicles and a very small amount of intervesicular tissue and blood (Leitz obj. 6, oc. 2, Tube 170). PLATE VI. FIG. 5. Showing cystic portion with bony mass, wall of haemorrhagic space and hyperplastic vesicles with copious blood supply (Leitz obj. 6, oc. 2, Tube 170). FIG. 6. Showing myxoedematous portion with much fibrous intervesicular tissue, large blood vessels and small atrophic vesicles (Leitz obj. 3, oc. 2). FIG. 7. Showing details of Fig. 6 (Leitz obj. 6, oc. 2, Tube 170). FIG. 8. Showing thick capsule and variable size of vesicles (Leitz obj. 3, oc. 2). FIG. 9. Showing capsule and small thick-walled vesicles of variable size (Leitz obj. 3, oc. 2). FIG. 10. Showing normal arrangement (Leitz obj. 3, oc. 2). ^^^ FIG. 11. Showing poor vascularity, intertubular spaces and many small vesicles (Leitz obj. 6, 0^2). FIG. 12. Showing normal arrangement, with good blood supply, small intertubular spaces, and large vesicles (Leitz obj. 6, oc. 2).

OURNAL OF EXPERIMENTAL BIOLOGY VOL. IV, PLATE V. FLg.3 Fig. 4 TESTIS AND THYROID IN HEN-FEATHERED SILVER-GREY DORKING COCK (pp. 73-80).

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. IV, PLATE Vr. hv FL&1O ANAN TESTIS AND THYROID IN HEN-FEATHERED SILVER-GREY DORKING COCK (pp. 73-80).