Variation in the Histological Condition of the Thyroid Glands of Sheep with regard to Season, Sex, Age, and Locality.

Transcription

1 Variation in the Histological Condition of the Thyroid Glands of Sheep with regard to Season, Sex, Age, and Locality. By Esther Lowe, M.Sc, Assistant Lecturer in Zoology, University of Manchester. With Plates 24 and 25. CONTENTS. PAGE 1. INTRODUCTION MATERIAL METHODS INFLUENCE OF SEASON ON HISTOLOGICAL STRUCTURE INFLUENCE OF AGE ON HISTOLOGICAL STRUCTURE INFLUENCE OF LOCALITY ON HISTOLOGICAL STRUCTURE INFLUENCE OF SEX ON HISTOLOGICAL STRUCTURE INFLUENCE OF CASTRATION INFLUENCE OF PREGNANCY SUMMARY 461 REFERENCES 463 EXPLANATION OF PLATES 463 INTRODUCTION. OUR present knowledge of the thyroid gland has been derived chiefly from the investigations of workers on the human gland. A comparatively small amount of work has been done on the thyroids of the lower animals, and this has been chiefly of an experimental nature, very little attention having been paid to the histology. In 1924, Mrs. Bisbee (Euth C. Bamber) (1), Lecturer in Zoology at the University of Liverpool, put forward an interesting suggestion as to the possible connexion between Braxy and thyroid activity in sheep.

2 438 ESTHER LOWE McCarrison (5, 1917) and others have demonstrated a seasonal prevalence of goitre in human beings, and Seidell and Fenger (10, 1912), by iodine estimations, demonstrated a seasonal variation in the activity of the thyroid glands of sheep, hogs, and cattle. It is very widely stated that a seasonal variation has been found in the glands of all animals investigated, but this statement appears to rest entirely on the work of Seidell and Fenger. Martin (8, 1912), however, working in the British Isles on sheep alone failed to find any definite seasonal variation ; also it is by no means clear that Seidell and Fenger's results were not complicated by sex variations which certainly exist and which were not taken into account. It seems by no means established, therefore, that seasonal variation does occur especially in the sheep of this country. In view of this uncertainty, Mrs. Bisbee suggested the present investigation into the activity of the thyroid glands of sheep, and its correlation with age, sex, season, and locality. Attention has also been paid to the influence of castration and pregnancy. The sheeps' glands were obtained from the Liverpool Abattoir, and my sincere thanks are due to Mr. Johnstone, meat inspector, and to Mr. Eoberts of Pritchard and Elsons, butchers, for their kindness in giving me every opportunity of obtaining material. The laboratory work was carried out in the Zoological Department of the University of Liverpool during the year October, 1924-October 1925, and I wish to express my thanks to Professor Dakin, Dr. Johnstone, and Mr. Horton of this University for their interest and suggestions during the course of the work. My especial thanks are due to Mrs. Bisbee for suggesting the work originally and for much valuable advice in regard both to the actual work itself and to the preparation of the paper. MATERIAL. The sheeps' thyroids used in this investigation were collected on Tuesdays or Wednesdays of each week so that the intervals between the successive samples were always regular. All the glands obtained were removed whole from freshly killed animals

3 THYROID OF SHEEP 439 and were in most cases placed in the fixative within minutes after the death of the animal, so that the risk of postmortem changes was as far as possible eliminated. The number of animals from which glands were taken varied from three to six per week according to the variety of material obtainable. When the sheep were all of the same age and sex, as was sometimes the case, it was not thought necessary to take more than three specimens, but more were taken on the occasions when a greater variety of material was obtainable. In all cases the approximate age of each sheep and the district from which it came were ascertained. The main difficulty, however, connected with this investigation was the impossibility of obtaining sheep of all kinds at regular intervals throughout the year. A regular supply of glands from young mature, uncastrated males was obtainable from October until the end of May. Young mature castrated males were easily obtained from October to the beginning of May; but during the summer months from May onwards, when the spring lambs were put on the market, it became impossible to obtain anything but male and female lambs. Throughout the year, the thyroids of mature females have been the most difficult to obtain regularly. During the autumn and early winter months, from October to January, mature females of all ages were as readily obtained as males and wethers ; but during the late winter and spring months, from January to May, not a single yearling female was obtained, although occasional aged females were put on the market. At the beginning of May the spring lambs were put on the market, but in addition to these, the supply of yearlings persisted throughout this month. These consisted of the yearling uncastrated males already mentioned and two yearling females which were obtainable on one occasion only. From the end of May onwards the glands of male and female lambs have been examined in about equal numbers to the complete exclusion of older sheep. This irregularity in the supply of material has, of course, added greatly to the difficulty of obtaining a correct estimate of the seasonal and sexual variation in the histoloeical structure

4 440 ESTHER LOWE of gland. At the same time, however, it emphasizes the need for an investigation of this kind, since the records of the seasonal variation in the iodine content of sheeps' thyroids carried out by Seidell and Fenger in America and by Martin in the British Isles, were derived from composite samples and no attention was paid to age or sex. In the case of the American sheep, however, variations due to locality were eliminated by including in each sample the glands of sheep from all parts of the country. METHODS. The glands were in all cases cut transversely into pieces before being placed in the fixative. In cases where only one or two portions were taken from one gland, the middle of the gland was always used, as it was thought that this would give the best estimate of the general condition of the gland. In cases where an enlarged isthmus was present, a portion of this was also fixed and sectioned. In all cases complete transverse sections were taken either from the middle portion of the gland, as mentioned above, or from various parts. During the course of this investigation many different fixatives have been used with varying degrees of success. Bouin's picro-formol was the first to be tried, but this was found to be quite useless for sheeps' glands ; it turned the colloid into hard stony masses which powdered on the microtome knife and completely ruined the sections. Flemming's fixative, which is recommended by Guyer for thyroid, was also found to be a failure. The colloid became so horny in consistency that the masses would not cut but came out of the acini whole. Carnoy's and Gilson's fixatives were both tried, but they both produced a serious shrinkage of the colloid. Finally, 10 per cent, formalin was used and was found very satisfactory. It fixes both cells and colloid satisfactorily and does not cause shrinkage. This fixative, therefore, has been used throughout the present work. The chief stains used have been Mann's methyl-blue-eosin and Mallory's triple stain. Iron haematoxylin has been used to a less extent.

5 THYROID OF SHEEP 441 THE THYROID GLAND OF THE SHEEP. INFLUENCE OF SEASON ON HLSTOLOGICAL STRUCTURE. Up to the present time none of the numerous workers on the thyroid gland appear to have published any account of the effect of season on the histological structure. The minute structure of the thyroid has been studied in connexion with temperature ; also several workers have devoted themselves to a study of variation in the iodine content of the gland, which is of course correlated with histological changes ; but the correlation between season and histological structure as such, has not, to the writer's knowledge, received any serious attention. It is known from McCarrison's (5, 1917) investigations that endemic goitre in man exhibits a definite seasonal prevalence. He states that in European countries goitre is supposed to originate most commonly in the months of March, April, May, and June. Also in parts of Himalayan India which are not reached by the monsoon, goitre tends to develop during the rainy season. Naturally the development of goitre' involves drastic histological changes, but McOarrison does not discuss the correlation of season with any particular type of histological structure ; he accepts seasonal variation of iodine content from the work of Seidell and Penger, and suggests that such seasonal variation in the iodine content of the gland may be correlated with the seasonal prevalence of goitre. Seasonal investigations of the iodine content of the thyroid glands of cattle, sheep, and hogs were carried out by Seidell and Fenger in America, and similar investigations for sheep only were made by Martin in England. In both cases the results were obtained by analysis of composite samples of the glands from large numbers of animals. In both cases the investigations were undertaken not with a view to studying a seasonal variation, but in connexion with an effort to fix a standard for thyroid preparations used in medicine. It was therefore of no interest to them to ascertain the sex of the animals whose glands were used, and the variation found in the iodine content was attributed to the influence of seasonal changes without taking into

6 442 ESTHER LOWE account the possibility of a seasonal variation in the proportions of the two sexes obtained. As it is well known that the iodine content, size, and physiological activity of the thyroid varies considerably with the sex of the animal, the predominance of one sex or the other amongst available material at different seasons of the year may have influenced considerably the results obtained. Be that as it may, the fact remains that the results obtained in England by Martin (8, 1912) differed widely from those obtained in America by Seidell and Fenger (10, 1912). Martin's results showed very little variation throughout the year. The lowest iodine content obtained was 0-3 per cent, of dry weight, and this minimum was obtained twice during the year, in the middle of November and again in the middle of April. The highest iodine content was 0-4 per cent, of dry weight, and was obtained in the middle of July. The estimate for October 14 was also nearly as high as that for July. Thus it is seen that the variation throughout the year in English sheep was less than 01 per cent. The results of Seidell and Fenger for Amefican sheep exhibit a strong contrast to this. The figures obtained by them show a marked seasonal variation. The iodine content altogether is considerably lower than that of the English sheep, the highest figure obtained being per cent, of dry weight on July 7, and the lowest, per cent, and per cent, obtained respectively on March 3 and May 26. As may be seen from the graph made by Seidell and Fenger (10, 1912), the curve drawn from the average results reaches its highest point early in November, and is at its lowest about April 14, and the figures show that the average iodine content for the months June to December is roughly three times that for December to May. It was also found that the weight of the gland varied in inverse proportion to the amount of iodine it contained, the average weight of the sheep's thyroid for summer and fall being 5-5 gm. and that for winter and spring 8-5 gm. A similar seasonal variation was also found in the iodine contents of the thyroid glands of cattle and hogs. In the case of the hog the maximum was reached early in September and the minimum early in March, whereas the maximum for cattle

7 THYKOID OF SHEEP 443 occurred early in October and the minimum early in February. The present investigation has been devoted to the study of the histological structure alone, and this has been used as an indication of the activity of the glands. The iodine contents have not been estimated, but the weights of the glands have been recorded for many weeks in succession at different periods of the year. The facts with regard to seasonal variation have been deduced chiefly from a study of the glands of young uncastrated males aged from 3 to 16 months, although both castrated males and females have been obtained over sufficiently long periods to give significant data. The uncastrated males include : (1) sheep, aged from 8 to 15 months, obtained during the period October- April (inclusive) ; (2) lambs, aged 3 months, and sheep aged months, during May ; and (3), lambs, aged from 4 to 7 months, during the period June-August (inclusive). It is fully realized that differences of age may have affected this account of seasonal variation to some extent. The results, however, do not seem to favour this conclusion, and since sheep are only born at one period of the year, viz. during January and February, it was impossible to obviate this difficulty. For convenience the results will be discussed in this section under the periods October-December, January-May, and May- August. October-December (inclusive). The investigations were begun early in October and for the first three months, October, November, and December, all the glands obtained from first-year males, both castrated and uncastrated, were found to be in a fairly uniform condition. Practically all the sheep obtained during this period were from North Wales, from the area including Euthin, Mold, Tallycefn, Abergele, and Trefnant, the latter place being the most common source of supply. There were, however, two uncastrated males which came from Yorkshire and whose glands were obtained on October 14. The glands of these two males formed the only

8 444 ESTHER LOWE two exceptions to the account given below for the period October-December and will be discussed later. The glands of all the other uncastrated males, and most of the castrated ones (wethers), contained a very large amount of colloid (figs. 1 and 8, PI. 24). The acini were greatly distended, until in many cases the epithelial cells were flattened and in some examples almost obliterated. There were, however, various intermediate stages in which the epithelium was of a low cuboidal character. The glands as a whole were characterized by complete absence of new secretion. There was a strong tendency to thickening of the intervesicular parenchymatous tissue accompanied by an increase in the number of fibroblasts. In many cases there was considerable evidence that a previous hypertrophic process had been subjected to some influence which had caused it to revert to the colloid state forming the so-called ' colloid goitre '. For instance, the colloid in many cases contained a considerable amount of cell debris, and, in the larger acini, plications of the lining epithelium were found. In many cases these glands possessed a large well-developed isthmus whose structure also exhibited characters of a colloid goitre and which was frequently the seat of a tumour which will be described in a later paper. The most advanced case of goitre was obtained on November 18 from a wether aged about 10 months. In this gland the parenchyma had become greatly thickened, but there was a varying amount of colloid and some of the acini were quite large. The parenchyma was opaque and appeared as an almost structureless mass containing an increased number of fibroblasts. The epithelial cells were mostly cuboidal in shape and there was evidence of a process of cell division the products of which had broken down and been cast into the colloid. The condition was also complicated by the presence of the tumour mentioned above. The only gland which showed any noticeable amount of new secretion during this period was that of a wether obtained on December 2. This gland agreed with all the others in that its

9 THYROID OF SHEEP 445 acini were greatly distended with large amounts of colloid. It also showed an extensive development of the fibrous stroma between the acini. Sprig-like projections from the acinar walls and a considerable amount of cell debris in the colloid showed it to he a gland which had undergone a period of hypertrophy. In all these characters it agreed with all other glands obtained during the same period, but it differed in that its epithelium was cuboidal instead of flat and in that large amounts of new secretion were still being poured into the already distended acini. McCarrison (6, 1925) records a similar condition arising in the glands of young animals under experimental conditions. He describes it as ' hypertrophic goitre, a balanced exaggeration of all the normal features of the gland's cycle of functional activity '. Only one other similar case has been found during the year, and that was the gland of a castrated yearling obtained on May 13. It is perhaps necessary to mention here that the glands of young females which were obtained regularly during the period October-December inclusive differed widely in their histological structure from those of the uncastrated males and wethers obtained during the same period. With one exception, which was the last of the series, they were all in a state of active secretion (fig. 2, PI. 24). It is not, however, proposed to discuss these in detail here as they will be dealt with later, in the section devoted to sex. The influence of age on the histological structure of the gland will also be discussed in a separate section. The weights of all the thyroids used during the months October-December were recorded and compared. The average weight of the glands of the first-year uncastrated males during this period M'as 2-34 gm. Those of castrated animals of the same age averaged 1-72 gm. and of females 1-98 gm. January-early May. The glands of the yearling uncastrated males and wethers during the following four months, January, February, March, and April were not nearly as uniform in character as those NO. 291 O g

10 446 ESTHER LOWE obtained during the period October-December. In early January the colloid condition characteristic of the autumn months tended to persist, but later in the month some of the males showed a more active condition of the gland. For instance, two males and a wether obtained on January 20 all showed signs of a return to the active condition. In one of the males the gland showed a considerable degree of hypertrophy. The acini contained a variable amount of colloid and the epithelial cells lining the acini were vacuolated and varied from a cuboidal to a columnar condition. One or two large acini in which the colloid condition still persisted were present; but on the whole the gland showed evidence of extreme activity. There was a plentiful supply of new secretion with a reduction in the amount of stainable colloid. The fibrous stroma was also well developed. The thyroid of the wether obtained on the same day showed an intermediate condition, the middle of the gland being more or less in the colloid condition and the outer regions being more active. This arrangement was an almost invariable rule in glands in which two different conditions coexisted ; the more centrally situated acini were always the less active. All the glands obtained in the following week, i.e. the last week in January, were in the colloid condition and showed no signs of recent activity. The gland of the one wether obtained was distinctly goitrous in condition. It contained smaller amounts of colloid than those of the males taken on the same date, but the connective tissue was very much thicker and showed an extensive development of nbroblasts. The glands of all the yearling uncastrated males obtained during February, March, and April showed signs of increased activity (fig. 6, PI. 24), in fact the colloid condition was not met with again in uncastrated male yearlings until May 13. The degree of activity in the glands during February, March, and April varied considerably, but all contained a fair amount of new secretion. The acini were more or less reduced in size, and the epithelial cells, instead of being flat, varied in shape from high cuboidal to high columnar and became vacuolated.

11 THYROID OF SHEEP 447 This increase of activity in the glands was accompanied by a marked distension of the capillaries. One or two extreme cases were obtained on February 4 and 18 in which the increased activity of the gland had caused hypertrophy. The epithelium in these cases was high columnar and showed evidence of active cell division. The acini were reduced in size and many of them were irregular in shape owing to plications of the lining epithelium. The glands of the castrated animals (wethers) during the same period, January-May 13, also showed a tendency to increased activity (figs. 7 and 9, PI. 25), but they were not as consistent as those of the uncastrated males, and although the majority were in an actively secreting condition, colloid glands without a trace of new secretion occurred sporadically throughout the period. One of these colloid glands was obtained on February 4, those of the uncastrated males obtained on the same date being in an active condition. Another was obtained on March 5, and was one of three glands taken from wethers of the same age and from the same district, the other two being in a very actively secreting condition. Two more were from sheep killed on April 22. One of the glands weighed 601 gm. and was the heaviest gland obtained during the year. The other gland appeared to be in an inert condition with small acini and no new secretion. From April 22 to May 10 the glands of all the yearling wethers were secreting actively and all showed a tendency to hypertrophy (figs. 7 and 9, PI. 25). One gland obtained from a wether on May 13 was in the colloid condition, but as this was the last date on which wethers were obtainable, it was impossible to carry this line of investigation any further. This was disappointing because, in the case of the uncastrated males, the beginning of May proved to be another turning-point in the seasonal variation. No glands were weighed during the spring months until March 11. From this date onwards the weights were recorded consecutively for castrated males alone. The average weight for castrated yearlings during the period March 11 to May 13 Gg2

12 448 ESTHER LOWE was 2-96 gm. This was considerably heavier than the average for the autumn and winter months. It was one of the glands during this period which weighed 6-01 gm. and, as noted above, was the heaviest gland obtained during the year. Early May middle of August. From May 6 onwards male and female lambs aged 3 months and over were obtainable, the males amongst the spring lambs being in all cases uncastrated. In addition to these the supply of yearling uncastrated males persisted throughout May, and on one occasion mature females were obtained. This was fortunate because the condition of the glands of these yearlings proved that the renewal of the colloid condition of the gland found in the younger lambs at this period was a definite seasonal change and was not due entirely to the difference in the age of the animals whose glands were used from the beginning of May onwards. On May 13 the glands of three uncastrated yearling males were obtained, two of which were in the colloid condition, while the third was very actively secreting (fig. 10, PI. 25). On May 27 the glands from four yearlings, two uncastrated males and two females, were obtained, and all were found to be in the colloid condition (figs. 11 and 12, PL 25). The gland of one of the females was a diffuse colloid goitre, the amount of colloid being so great that some of it had forced its way out of the acini and lymph channels and had formed large translucent globules in the gland, which were clearly visible to the naked eye..evidences of previous hypertrophy were present in the gland (fig. 11, PL 25). The first spring lambs, aged from 2 to 3 months, were obtained on May 6, and an examination of their glands showed that these were also in the colloid condition, with greatly distended acini lined by flat epithelium. On May 20 the glands of five spring lambs, two females and three males, were obtained, and on this occasion it was found that the glands from the females were actively secreting, whereas those of all the males were in the colloid condition. One of the female thyroids was very active

13 THYROID OF SHEEP 449 and exhibited a certain amount of hypertrophy, but the other was active in certain areas only, the rest of the gland being in the colloid condition with acini of moderate size lined by flat epithelium. From the beginning of May until the middle of August the glands of all the male lambs were found to be in the colloid condition, and many of them showed evidence of some degree of previous hypertrophy. The glands of female lambs examined during the same period were not as uniform in appearance as those of the males. All contained large acini, but the amount of new secretion varied considerably. In some cases the epithelium was cuboidal and new secretion was entirely absent. In the cases in which the gland was producing new secretion, this activity was not as a rule associated with any diminution in the amount of older colloid. Many of the glands had cell debris in the colloid and the acinar walls were several cells thick, suggesting previous hypertrophy. Some of the glands were of a mixed character, the more centrally situated acini being in the colloid condition, while the outer areas of the gland were more actively secreting. All the glands obtained between May 6 and July 8 were weighed and the averages calculated. It was found that in the cases of lambs the glands of females were heavier than those of males, the average weight for male spring lambs being 2-37 gm. and that for females 2-43 gm. The average weight for yearlings during May was greater than that for lambs, the average weight for uncastrated males being 3-78 gm. and that for females 2-55 gm. A comparison of these weights with those obtained for the autumn and early winter months shows that the glands of both sexes are heavier during May and June than during October, November, and December. The increase is obviously not due entirely to a difference in age as the glands of 4-monthold lambs in June were heavier than those of 9- or 10-monthold lambs in November and December. A summary of the results obtained shows that there is a definite seasonal variation in the condition of the glands of young uncastrated males. The colloid condition of the gland

14 450 ESTHER LOWE which prevailed during the autumn and early winter months was replaced at the end of January by a more active condition which continued until May, when it gave way once more to the colloid condition. The glands of castrated animals were only obtainable during the period October to May, but during this time their condition was more or less in agreement with that of the uncastrated males. They showed the same seasonal variation, but during the spring months when the glands of the young uncastrated males were all actively secreting, those of the wethers were not as uniform in condition and a few colloid glands were obtained. The glands of young females were only obtainable at two seasons of the year, viz. the autumn and again during the summer months May to August, 1 but a striking difference was observed in the glands obtained during these two periods. During the autumn the glands were all actively secreting and showed a reduction in the amount of old colloid accompanied by a tendency to hypertrophy. During the summer months, on the other hand, the glands contained very large amounts of colloid and the acini were greatly distended. In fact the glands of females, both spring lambs and yearlings, during this period, differed very little in condition from those of the males. The main difference observed was that the epithelium was not as flat in the females as in the males, and many -of the female glands were producing a certain amount of new secretion in spite of the presence of large amounts of colloid. It is of interest to notice in this connexion that the return of the glands of uncastrated male sheep to the colloid condition at the beginning of May coincided with a great increase of temperature which took place throughout the British Isles at that time. From the experiments of Mills (9, 1918) with the glands of guinea-pigs, it is known that an increase of temperature induces the colloid condition, whereas a decrease of temperature causes increased activity. This would provide a satisfactory explana- 1 The glands obtained during the autumn were from mature females aged 8-11 months. Those obtained during the summer months were from lambs aged 3-7 months, with the exception of two which were obtained in May from mature females.

15 THYROID OF SHEEP 451 tion of the prevalence of the colloid condition in the glands of both sexes during the summer, but it does not explain the occurrence of the colloid condition in the glands of all the Welsh males during the autumn and early winter months, for this period included some of the coldest days of the year. The active condition of the glands of female sheep during this period does agree with the observations of Mills for guinea-pigs, but in the case of males, at any rate, the seasonal variation does not seem to depend entirely on changes of temperature. INFLUENCE OF AGE ON HISTOLOGICAL STRUCTURE. Most of the evidence in regard to the effect of age on the histological condition of the thyroid seems to have been derived from the study of the human thyroid, and the accounts given by the various workers are not entirely in agreement; for instance, McCarrison (5, 1917) states that after the age of 40 in human beings the gland tends to atrophy, the epithelium becomes less active, and its colloid and iodine content decreased. In opposition to this we have the account given by Vincent (12, 1922), who states that the maximum iodine content in the thyroid of man is found between the ages of 40 and 60 years. In a later paper McCarrison (6, 1925) classifies goitre into three types and states that the incidence of acquired hyperplastic or parenchymatous goitre is low in early childhood, but increases gradually with advancing years ; whereas colloid goitre has its highest incidence in early life, tending to disappear after full maturity has been attained. The remaining type, the hypertrophic goitre, described as an early stage of the parenchymatous goitre, tends to arise in young animals under insanitary conditions. Marine and Lenhart (7, 1909), on the other hand, state that the colloid condition occurs in older animals and active hyperplasias in young animals. Nothing is known about the effect of age on the histological structure of the sheep's thyroid, but in 1913 Sutherland Simpson (11) published an account of some interesting experiments which he carried out on the relation of age to the effects of

16 452 ESTHER LOWE thyroidectomy in sheep. It was found that the removal of the thyroids and internal parathyroids from 2-year-old sheep had no deleterious effects on the animals provided they were kept under hygienic conditions. The same operation was performed on 6-7-month-old lambs and they also suffered no ill-effects, but 2-month-old lambs developed into typical cretins as a result of the operation, and lambs aged from 5 to 7 weeks developed tetany and died. These experiments suggest that the thyroid is a much more important organ in the young lamb than it is in the adult sheep. In the present paper an attempt has been made to correlate age with thyroid activity. The results so far discussed in connexion with seasonal variation have included only the young sheep aged from about 4 to 16 months, but in addition to these older animals were obtained periodically until May 6. Uncastrated males, females, and wethers, all older than 21 months, were obtainable on various occasions, but not in sufficient numbers nor at sufficiently regular intervals to enable one to form any estimate of a seasonal variation in their thyroids ; the ages of these sheep ranged from 1 year 9 months to 4 years and over. From the foregoing section it is clear that in all young animals the thyroid goes through alternate periods of activity and rest, and that these changes are correlated with season. In older animals, on the other hand, there seem to be no such obvious seasonal changes. During October and November the glands of four old sheep were obtained, two from wethers aged 1 year and 9 months and two from 4-year-old females. In contrast to the glands of the younger animals obtained during the same period, none of these four glands was completely in the colloid condition. In the glands from the two castrated males the acini were very irregular in size, some containing a moderate amount of colloid, others practically none. There was a small amount of new secretion and the epithelial cells were more or less cuboidal. The connective tissue was thickened and one gland was distinctly goitrous in character. Both glands contained tumours,

17 THYROID OF SHEEP 453 one small and the other large. The glands of the females were less goitrous than those of the wethers and contained rather more colloid. The connective tissue was not abnormally thickened and the epithelial cells varied from a cuboidal to a low columnar shape. The amount of new secretion in both cases was small, but groups of cells were present in many of the acinar walls, particularly in the outer regions of the gland where the acini were small. The heavier of the female glands contained a large tumour. During January and February the glands of seven older animals were examined. On January 13 the glands of three castrated animals of different ages were obtained. The ages were 2 years, 2 years and 9 months, and 3 years respectively. The gland of the 2-year-old animal was in the colloid condition with greatly distended acini and flattened epithelial cells. It was practically devoid of new secretion and contained a large tumour. The gland of the animal aged 2 years and 9 months was also in the colloid condition, but the epithelium in this case was low cuboidal instead of flat and there was no tumour. In some parts of the gland there was evidence of previous active cell division. The gland of the 3-year-old animal was in a condition of active hypertrophy. The size of the acini was variable throughout the gland. In some areas colloid was almost absent; and the epithelial cells in most areas were in a state of active cell division. February 10 was the next date on which glands were obtained from aged animals. On this date the glands of four females, three of which were pregnant, were obtained. All were found to be in the colloid condition with flat acinar epithelium and no new secretion. The connective tissue was greatly thickened in all four cases, but was more opaque and goitrous in appearance in the pregnant than in the non-pregnant animals (cf. figs. 4 and 5, PI. 24). In the latter the intervesicular parenchymatous cells were not as completely atrophied as in the pregnant females. The glands of the pregnant females all contained tumours. No more glands from aged animals were obtained after this date until April 29, when glands were taken from an

18 454 ESTHER LOWE aged female and a 3-year-old wether. That of the female was in the colloid condition throughout. The epithelium was completely flattened and the acini were fairly regular in size and shape and all greatly distended with colloid. Traces of previous hypertrophy were present in the form of localized thickenings of the connective tissue. The gland of the 3-year-old wether (fig. 8, PI. 25) was also mainly in the colloid condition with signs of recent hypertrophy in the form of infolded acinar walls and areas of cell multiplication. In one area the hyperplastic condition still persisted. The epithelium throughout was mainly low cuboidal. In the following week the last of the series of glands from aged animals was obtained. This was from a very old male whose age could not be definitely determined. The gland was one of the heaviest obtained, its weight being 5-12 gm. It was in the colloid condition throughout with greatly distended acini of irregular size. The intervesicular tissue was greatly thickened and invaded by fibroblasts. In many of the larger acini the epithelium was plicated in places and the colloid contained cell debris from an earlier hyperplastic condition of the gland. A very large tumour was present. Viewed as a whole, these results seem to point to the conclusion that the colloid state is the one most commonly found in the glands of old sheep, irrespective of sex or season, although it is fully realized that the glands examined were too few to admit of any but the most tentative of generalizations. The fact, however, that with one exception all the glands of aged animals obtained between January 13 and May 6 were in the colloid condition, whereas during this period those of the young animals obtained were found to be actively secreting, does suggest that the apparently permanent colloid condition is directly related to the advanced age of the animals. The colloid condition in young animals after its disappearance in early January did not appear again until the beginning of May. With increasing age, therefore, the gland appears to lose its power of response to environmental changes.

19 THYROID OF SHEEP 455 INFLUENCE OF LOCALITY ON HISTOLOGICAL STRUCTURE. Most of the sheep used in this investigation have been obtained from North Wales, from the area including Denbigh, Euthin, Mold, Abergele, Wrexham, Corwen, and Trefnant. The only exceptions were three sheep (two males and one female), obtained from Yorkshire, and eleven spring lambs from Ireland. The exact district from which the Irish lambs came was not known. Mention has already been made of the fact that the glands of the Yorkshire sheep differed from those of the Welsh sheep obtained during the autumn months. Whereas the glands of the Welsh uncastrated males during this period were all in the colloid condition with a strong goitrous tendency, those of the two uncastrated males from Yorkshire were in a much less extreme condition. The acini were very varied in size, but none of them were as large as those of the Welsh males. Also there was a certain amount of new secretion and in some regions the capillaries were somewhat dilated. In fact, both showed a moderate amount of activity. The gland of the Yorkshire female was in a similar condition to that of the males and was not as active as those of Welsh females. None of the glands showed evidence of hypertrophy. It is of course impossible to draw any conclusion on the evidence of three glands, but their condition is recorded because they showed an interesting variation from the general rule, which was possibly due to a difference of locality. The only other exceptional gland obtained during the same period was that of a Welsh female obtained on December 9 from Abergele. This gland, instead of being in the actively secreting condition which was characteristic of all other females of the same age, was in the colloid condition, with distended acini lined by flat epithelium and without any trace of new secretion. It showed no signs of previous activity. The condition of this gland is interesting in view of the fact that this was the only occasion on which sheep were obtained from a place near the sea-coast, and it is known from the records of various workers

20 456 ESTHER LOWE on the thyroid gland that there is a higher iodine content and less activity in the glands of animals living near the sea-coast, due probably to an increase in atmospheric iodine. Hunter and Simpson (4, 1914) estimated the iodine content of sheep pastured in the Orkney Islands and found that the glands contained a very high percentage of iodine, in some cases about three times the normal amount. The glands of the Irish lambs obtained during June and July showed no difference in either sex from those of the Welsh lambs obtained at the same time. As the part of Ireland from which they came was not ascertained, it was impossible to say whether they had pastured in a similar environment to that of the Welsh lambs or whether the similarity was due to the fact that the seasonal influence in this case was stronger than any local influence. INFLUENCE OF SEX ON HISTOLOGICAL STRUCTURE. It is well known from the work of McCarrison (5, 1917) that in man the thyroid -gland of the female is larger, more active, and contains more iodine per unit of body-weight than that of the male. Eenger (3, 1914) also states that in cattle the females contain more thyroid tissue and more iodine therein per unit of body-weight than the males. It is also known that after sexual maturity is attained in human beings endemic goitre is more common in females than in males, although up to the age of puberty it affects both sexes equally. The present investigations have shown that in sheep there is a very definite difference in the condition of the gland in the two sexes during the months October, November, and December (cf. figs. 1 and 2, PI. 24). All the glands obtained from yearling uncastrated males during this period were in the colloid condition, with the exception of two, which were from a different district, but the glands of yearling females during the same three months were all in a very actively secreting condition. The acini were diminished in size and there was a reduction in the amount of old colloid. The epithelium lining the acini was columnar, whereas in the males it was flat. In practically all the females

21 THYROID OF SHEEP 457 it showed some degree of hypertrophy either in the form of cell multiplication or by the folding of the epithelium into the colloid in some of the acini. The only gland whose condition did not conform to the description given above was that of the female obtained on December 9 from Abergele. This gland has already been discussed in the section on the influence of district. Its condition resembled that of the males obtained on the same date much more than any of the females obtained during the same period. In addition to the difference in structure of the glands of the two sexes, there was also a difference in the average weights, that for yearling uncastrated males being 2-34 gm. as compared with 1-98 gm. for yearling females. The failure of the supply of thyroids of young females during the late winter and spring months made it impossible to estimate the sex difference during this period. It has already been stated in the section dealing Avith the effects of age that there was no sex difference in the glands of older animals during this period. During the summer months, May, June, July, and August, females were again obtainable, although with the exception of those obtained from yearlings on May 27, all the glands examined during this period were from young lambs aged from 4 to 7 months and which therefore were not sexually mature. The glands of the four yearlings, two uncastrated males and two females, obtained on May 27, have already been described in the section dealing with the effects of season on histological structure. Portions of two of them are represented in figs. 11 and 12, PI. 25. All were in the colloid condition and showed evidence of having previously undergone a period of activity. This was particularly evident in the case of one of the females. This gland, which was the lightest of the four, exhibited an extreme condition of diffuse colloid goitre. The acini and lymph channels were greatly distended, and in addition much of the colloid had escaped and was lying free in the gland, enclosed only by the surrounding connective tissue capsule, and when the gland was cut it poured out as a viscous fluid. An examination of the sections showed that throughout the gland the acini

22 4.58 ESTHBK LOWE were invaded by plications of the lining epithelium whose cells were flattened. Cell-masses were present in the epithelium of some of the acini and the colloid contained desquamated masses of cell debris which had been cut off from the lining epithelium. The cells were not uniformly flat throughout the gland, although this condition was most common, but in some of the very large acini the cells retained their columnar shape in spite of the amount of colloid present, and some of them were still producing globules of new secretion. The gland of the other yearling female was also in the colloid state, but its condition was not nearly so extreme. The colloid condition was most pronounced in the middle of the gland, tending to give way to a more active state in the outer regions. The glands of the two uncastrated male yearlings were purely colloid in condition (fig. 12, PI. 25), and showed no trace of new secretion. The acinar epithelium of both showed evidence of previous activity in some parts of the glands. The glands of the males were distinctly heavier than those of the females. They weighed 4-11 gm. and 3'29 gm. respectively, whereas the females only weighed 2-87 gm. and 2-73 gm. The glands of the male and female lambs obtained during May, June, July, and August were also with very few exceptions in the colloid condition, but whereas those of the males were all pure colloid glands throughout and contained practically no new secretion, most of the glands from females were producing a certain amount of new secretion in some parts if not throughout, in spite of the fact that the acini were quite as large and contained as much colloid as those of the males. The acinar epithelium was flat in the glands of most of the males, but in the females it was more often cuboidal, although a certain amount of variation occurred in both. This difference in the condition of the acinar epithelium was the only difference observed in the glands of the two sexes during the summer months, but as the animals from which the glands were obtained were not sexually mature, one would perhaps scarcely expect to find a very striking difference between the sexes. The glands of the very few yearlings obtained during May agreed with those

23 THYROID OF SHEEP 459 of the lamhs in showing very little difference between the sexes, but as only two females and five males were obtained it is impossible to draw any reliable conclusions. The average weight of the thyroid of female lambs for the four months under discussion was 2-56 gm. as compared with 2'37 gm. for males. The two female yearlings averaged 2-55 gm. and the five males 3-78 gm. Thus it is clear that a marked difference in the histological structure of the glands of the two sexes was found only during the autumn and early winter months. The condition of the females during the spring months was not recorded, but during the summer months very little difference was found in the glands of the two sexes, either in spring lambs or in yearlings. INFLUENCE OF CASTEATION. Practically nothing is known of the effect of castration on the human thyroid ; but Fenger (3, 1914), in his investigation of the iodine content of the thyroid gland of cattle, found that castrated males contain less thyroid tissue than either uncastrated males or females, and that the iodine content per unit of body-weight is intermediate between that of the uncastrated male and the female. With regard to the amount of thyroid tissue in castrated animals, the present results are entirely in agreement with those of Fenger. All the thyroids obtained during the months October, November, and December were weighed and the averages calculated. It was found that the thyroids of the castrated animals were lighter than those of either the males or the females, averaging only 1-72 gm., whereas the glands of females averaged 1-92 gm. and those of the males 2-34 gm. During the spring months only a proportion of the glands obtained each week were weighed, but these showed a similar difference in the average weights of castrated and uncastrated males. The average for the uncastrated males was 3-5S gm., and that for the wethers, 2-86 gm. With regard to the histological structure, the glands of wethers in most cases seemed to follow fairly closely the

24 460 ESTHER LOWE seasonal variations observed in the glands of uncastrated males, except that during the early spring months, when the glands of all the males changed from the colloid to the actively secreting condition, the glands of the wethers were much more varied in their condition. Some became actively secreting, like those of the uncastrated males, while others remained in the colloid condition. INFLUENCE OF PREGNANCY. Most of the workers on the human thyroid state that an enlargement of the gland usually occurs during pregnancy. McCarrison has observed that goitres commonly originate during pregnancy and that enlargement of previously existing goitres is particularly liable to occur during this period. Blair Bell (2, 1920) states that thyroid enlargements during pregnancy are due to storage of colloid. Very little is known about the effects of pregnancy on the thyroids of the lower animals, but Fenger, from his investigations on the thyroids of cattle, finds that there is no apparent difference in size and physiological activity between glands from pregnant and non-pregnant females. During the present investigation glands from pregnant females were only available on one occasion. The glands of three aged pregnant females were obtained on February 10, and on the same date the gland of another non-pregnant aged female from the same district was also taken. All the glands were found to be in a condition of colloid goitre with thickened intervesicular tissue and abundant evidence of previous hypertrophy (figs. 4 and 5, PI. 24). Those of the pregnant females, however, were more goitrous in structure than the gland of the nonpregnant animal. In the latter the cells of the intervesicular tissue were still distinguishable, whereas in the glands of the pregnant animals many of the cells appeared to have atrophied. The glands of the pregnant animals weighed 3-42 gm., 2-67 gm., and 2-6 gm. respectively. They were all heavier than the gland of the non-pregnant female, which weighed only 2-14 gm. This is of course very slight evidence, but as far as it goes it