6I2.6I6.I: necessity for a uniform diet and for the elimination of infection has not. Surgeons, Columbia University, New York City.

Transcription

1 1 6I2.6I6.I:612.4 THE EFFECT OF GONADECTOMY ON THE ADRENAL, THYROID, AND PITUITARY GLANDS. BY DOROTHY H. ANDERSEN AND HELEN S. KENNEDY. (From the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York City.) INTRODUCTION., IN view of the recent increase in our knowledge of the intimate relation between the endocrine glands and reproduction and the contradictory reports in the literature as to changes produced in the endocrine glands by removal of the reproductive organs, it has seemed to us advisable to sift the literature critically and also to obtain new experimental data. The necessity for a uniform diet and for the elimination of infection has not been fully appreciated in most of the earlier work, and the recent application of the statistical method to biological data has brought greater certainty as to the significance of variations from the normal under different experimental conditions. Our observations on the changes in the pituitary and adrenal glands associated with cestrus have provided us with more satisfactory information concerning normal adult female rats than has previously been available. I. THE ADRENALS. (1) Review of literature. The literature on the effect of gonadectomy upon the adrenal gland is confusing, partly because of the variety of results and partly because of the inadequate data in most of the reports. Very few of the workers escaped one or another of the errors inherent in work with the adrenal gland, especially those resulting from disregard of the following points: (1) the wide normal variability in the weight of the adrenal gland necessitating the use of many animals; (2) the changes which accompany various phases of reproduction, as puberty, cestrus, and pregnancy; (3) the age and body weight of the animals used; (4) the presence of accidental infections; (5) the effect of diet; (6) the interval between operation and the sacrifice of the animal. PH. LXXIX. 1

2 2 D. H. ANDERSEN AND H. S. KENNEDY. If the various conditions of the experiments be disregarded for the moment, the conclusions of the authors as to the effect of gonadectomy on the adrenals may be summarized as follows: (1) Hypertrophy was found: in male rabbits by Schenk [1910], Kolde [1913], Zchwetadse [1927], Leinati [1929], and Livingston [1916]; in female rabbits by Schenk, Kolde, and Leinati; in male guinea-pigs by Leinati and Rigano-Irrera [1929]; in female guineapigs by Raineri [1908]; in male rats by Hatai [1915]; in male mice by Altenburger [1924], Masui and Tamura [1926], and Deanesly [1928]; in dogs by Feodosyeff [1905]; and in frogs by Ciaccio [1903]. (2) Transitory hypertrophy was found: in male and female rabbits by Soli [1909]; in male guinea-pigs by Marrassini [1906]; and in guineapigs by Soli. (3) No change was found: in male rabbits by Marrassini and Luciani [1911]; in female rabbits by Marrassini and Luciani, Dick and Curtis [1912], or by Livingston [1916]; in male and female guinea-pigs by Marrassini and Luciani; in immature female mice by Deanesly [1928]; in mature female mice by Howard-Miller [1927]; in male and female dogs by Pianese [1929]; and in male sheep, oxen and capons by Marrassini and Luciani [1911]. (4) Slight atrophy was reported in female rats by Hatai [1915]; in female mice by Masui and Tamura; in male rats by Minowada [1928]; in adult female mice by Deanesly [1928]; and in capons by Soli [1909]. A critical survey of the work on each species is presented. (1) Rabbit. This is the species most often studied. The earliest report on this animal which we have found is that of Soli in In the course of his work on the effect of castration on the thymus, he castrated seven male and spayed three female rabbits with ten controls paired by age and presumably by sex, and killed 1-6 months after operation. He gave no weights, but stated that the adrenal volume was increased in those killed 1 month after operation and decreased slightly in those killed after longer intervals. Schenk in 1910 spayed eleven female rabbits, with four controls: four were spayed at the ages of 3-7 weeks with one control, and killed after days; three were spayed at the age of 6 months with two controls and killed after 60 days; and four at the age of 9 months or over, with one control, and killed after an interval of days. One adult male without a control was also castrated. Schenk gave no body or adrenal weights, but made a histological study of the adrenals and

3 EFFECT OF GONADECTOMY ON GLANDS. reported uniform results, with hypertrophy of the cortex, enlargement of the cells of the zona fasciculata, and a slight decrease in the size of the medulla. The inadequacy of the controls and the lack of standardization cast doubt onthe conclusions. MarrassiniandLuciani [1911] castrated seven male and spayed six female rabbits of various ages with an equal number of controls paired by sex and weight. The animals were killed after intervals of days, and no constant change in the adrenals was observed, although the variations were great. Dick and Curtis [1912] spayed nine adult female rabbits with six controls, and killed them days after operation. They recorded the weights of the adrenals and of the eviscerated animals, both of which varied greatly, but found the same mean relative adrenal weight in each series, namely 0-32 mg. per g. of body weight. They made histological studies of the adrenals and found no difference between those of the two series. Kolde [1913] spayed eight females and castrated two males, at the ages of 3-6 months and killed them after 3-6 months. As controls he used five normal males, eleven non-pregnant females and five pregnant females of various ages from 4 months to several years. He gave no weights, but stated that sections of the adrenals of the castrated rabbits appeared larger than those of the normal ones. Livingston [1916] studied twenty-one male rabbits castrated at a body weight of g. with sixteen controls, and nine female rabbits spayed at a weight of g. with eleven controls. He killed these animals after intervals of days, when the average body weight was approximately 2 kg. He found a slight but irregular increase in weight in the adrenals in the castrated males, but no change in the spayed females as compared with controls of the same sex. Zchwetadse [1927] castrated five male rabbits and removed one testis from each of two more. He had one control. The ages were not reported, but the body weights at operation varied from 845 to 3825 g. The animals were killed at intervals of I-4 months. The adrenals were not weighed but were sectioned, and changes in both medulla and cortex were found in all cases. The cortical changes varied with the interval after operation, with a temporary increase and later a decrease in the size of the cells of the fasciculata. Leinati [1929] used six castrated rabbits with three controls and one spayed female with one control. Three of the males and two controls were 3-months-old litter mates, killed at intervals of 10, 20, and 30 days respectively. He reported cortical hypertrophy, but gave no body weights and gave adrenal weights in round numbers, so that their accuracy is open to question. The figures indicate some 3

4 4 D. H. ANDERSEN AND H. S. KENNEDY. hypertrophy. The two females were of the same litter, and after 40 days the spayed one had larger adrenals than the control. The remaining three castrated and one control male rabbits were adults, the interval was 5-6 months, and examination of the figures does not reveal the hypertrophy which he reported, if the body weight be considered. He also stated that accessory adrenals were hypertrophied and therefore easier to find in the castrated animals. Since no one of these workers has an adequate series at constant conditions of age and post-operative interval, and the only series in which the animals and adrenals were weighed (namely that of Marra s sini and Luciani [1911]) showed such extreme variations in the weights of the adrenals, we may conclude with these workers that no constant change has been demonstrated in the adrenal glands of the rabbit following gonadectomy. (2) Guinea-pig. The earliest experiments of the kind on the guinea-pig which have been found are those of Marrassini in He castrated twenty males with fourteen male controls, the body weights at death varying from 320 to 650 g. The weights are approximately parallel in the two series. He killed the operated animals at various intervals, four after 4 days, five after 8 days, three after 12 days, five after 15 days, and three after 24 days, weighed both the adrenals and the guinea-pigs and calculated the weight of the adrenal in percentage of that of the body. There was a marked increase in the relative weight for the first 16 days, with the highest weight at 8 days, namely *078 p.c., followed by a consistent decrease to *052 p.c. in the 24-day group. This is still higher than the controls which vary from to p.c. Histologically this hypertrophy was found due to an increase in the vacuoles and size of cells in the fasciculata. This is the longest as well as the most carefully controlled series reported on the guinea-pig. In 1911 Marrassini and Luciani noted the adrenal weights in the course of a series of experiments on the effect of castration on the hypophysis. In six castrated male and eight spayed female guinea-pigs with an equal number of controls paired by age and sex and killed at intervals of from 3 to 90 days, the adrenal weights, the ages of the animals and the postoperative intervals varied so greatly that no conclusions can be drawn. The authors include the figures in their tables without discussing them. Raineri in 1908 spayed a series of female guinea-pigs and killed them after intervals of 4 days to 3 months. He also hysterectomized other animals and performed the double operation on some. No mention of age, weight, or numbers of animals was made. He found a progressive hyper-

5 EFFECT OF GONADECTOMY ON GLANDS. trophy with a maximum value of 4x normal after 3 months. Without records of controls this figure appears unreliable, especially in view of Marrassini's more careful work. Soli in 1909 examined four castrated and four control guinea-pigs and reported the same findings as in his experiments with rabbits. The next experiments on guinea-pigs appear to be those of Rigano-Irrera in 1929, who castrated twelve and vasectomized twelve animals with twelve controls, all adults, and killed them at intervals of 48 hours to 350 days. The figures which he gives indicate an increase in adrenal weight during the first month, which is about equal in the castrated and vasectomized animals, and which later drops, but not to the level of the controls, and remains higher in the vasectomized than in the castrated animals. This is also true in the group killed after days. No note is made as to the age or weight of the animals at operation nor as to the age of the controls. Leinati [1929] castrated five young guinea-pigs with three controls, with an average weight for all eight of 350 g. He killed them at intervals of days and found cortical hypertrophy in all of them, due to hypertrophy of the zona fasciculata. The body weights varied greatly, but calculation of the relative weights from his figures shows what appears to be a definite hypertrophy, which, however, was more marked in those of his animals which were smaller and probably younger. Parhon, Marza and Kahane in 1926 reported the water content of the adrenals in ten castrated male guineapigs and their controls which were as near the same age as possible. The animals were sacrificed 1-10 months after castration. In seven pairs the water content was greater in the castrated animals, while in three the reverse was true. The authors concluded that there was a temporary decrease followed by an increase in the water content of the adrenals in the castrated animals. In a survey of the experiments on guinea-pigs one is impressed with the greater care in standardization found in the work of Marrassini, and with his more detailed report of the experiments. His animals are also the most numerous and are in groups of three to five under a given set of conditions, with careful notes as to organ weights. In the work of the other investigators the age of each animal at operation and the interval after operation varies so widely and the normal variations in the adrenal weight are so completely disregarded as largely to invalidate the results. The additional fact that M a r ra s s i n i's conclusions agree with those of the present authors tempts them to believe him. (3) Rat. Hatai in 1915 reported a series of experiments in rats, as follows: (1) twenty-six males were castrated at days and killed after days with twenty-seven controls; (2) five males were cas- 5

6 6 D. H. ANDERSEN AND H. S. KENNEDY. trated at days and killed after days with six controls; (3) eight males were castrated at days and killed after days with seven controls; (4) twenty-eight females were spayed at days and killed after days with thirty controls; (5) eight females were spayed at days and killed after days with nine controls; (6) seven females were spayed at days and killed after days with nine controls. Instead of comparing the adrenal weights directly, he compared them with the values given for rats of the same body length according to the Wistar Institute tables. He calculated the percentage difference of the adrenal weight of each rat from the adrenal weight given for rats of its body length in these tables. He then obtained the mean of these figures for the percentage difference of all the control rats and the corresponding mean for the castrated rats, and added these two figures to obtain the percentage difference between mean and control rats. This method ignores the possibility of variations in adrenal weight from causes other than those of sex, body length, and castration. No explanation is offered for the fact that the adrenal weights of the control rats differed as much from those given in the standard tables as did the adrenal weights of the operated animals. This difference may well be due to variations in diet, environment, season or degree of infection in his control rats as compared with the rats used for preparation of the tables. Direct calculations from his figures show a much smaller difference between the castrated and control animals in five of the six experiments, than does his method of calculation. The figures are given for comparison: Mean adrenal weight in g. A & 5 p.c. difference by p.c. change Series Castrated Control direct comparison (Hatai) * * The difference between the two methods of calculation is significant only in the male series. They are partly based on the fact that his control animals grew to a lesser length than the castrated ones, which is not the universal experience. The old adult controls of Series 6 have much smaller adrenals than the young adult controls of Series 5. This can be explained by our observations that old female rats often have irregular cycles, which we have found to be associated with low adrenal weights [An dersen and Kennedy, 1932]. The different results obtained in Series 5 and 6 can be interpreted as due to age changes in the controls of Series 6 rather

7 EFFECT OF GONADECTOMY ON GLANDS. than to age changes in response to gonadectomy. Hat ai concluded that the adrenals showed opposite reactions in the two sexes as the result of gonadectomy, with an increase in castrated males and a decrease in spayed females. He also removed one gonad from a series of males and females and found no changes in the adrenals. After ligation of the spermatic cords he found no adrenal changes unless the testes had undergone advanced atrophy, in which case the changes were identical with those found in the castrated animals. Minowada [1928] observed a slight decrease in weight in the adrenals of male rats after castration, but found no histological changes. (4) Mouse. The mouse has been the object of several studies. Altenburger [1924] found hypertrophy in the adrenals of four male mice castrated at 3 weeks and killed at about 4 months of age, as compared with two controls. Masui and Tamura [1926] castrated thirty mice and spayed thirty more at the age of about 20 days, and had an equal number of controls of the appropriate sexes. They killed them at the age of 90 days and sectioned the adrenals. They did not say that the sections were serial, but measured the square area of a section, presumably through the centre of the gland, with the width of the medulla, reticularis, and the remaining cortical zones. In the male castrates they described a decrease in the width of the fasciculata and glomerulosa, taken together, with an increase in the reticularis. In the females there was a slight decrease in size without structural changes. Howard-Miller [1927] reported no change in the x zone after ovariectomy in twelve mice in comparing one adrenal removed at the time of spaying with the other removed days later. Deanesly [1928] made a more careful study in a shorter series of fourteen castrated males with fourteen adult male controls and ten spayed females with fourteen adult female controls. The males were castrated at varying ages from 3 to 12 weeks and killed after 2-10 weeks. She found an increase in the adrenals up to 40 p.c. if the operation was performed before puberty, with the appearance of an x zone similar to that in the female; this zone is usually atrophic in the adult male. Five of the females were spayed before maturity and examined after 2 and 12 weeks. The adrenals resembled those of the normal adult female. In the five mice spayed after maturity the x zone had become partially degenerated at 16 weeks and completely so at 30 weeks after operation. These observations are based on serial sections, not on weights. These various authors are in essential accord as to the effect of gonadectomy on the adrenals in the mouse, namely a hypertrophy in the male, due chiefly to the increase in the x zone, more marked in operations on the immature animals, which 7

8 8 D. H. ANDERSEN AND H. S. KENNEDY. may be interpreted as continuation of the immature structure. There is little, if any, change in the female, in which the x zone is found in normal immature and mature animals. The presence of the x zone in this species makes comparison with other species difficult. (5) Dog. Feodosyeff [1905] reported the results of spaying in female dogs. The age, weight, number of animals, and existence of controls was not mentioned. He killed them at varying intervals up to 9 months and 3 weeks. He gave a minute description of the histological appearance of the adrenals, which consisted of marked hypertrophy of the zona glomerulosa and zona fasciculata with mitoses and increase in the size of the cells. Pianese [1929] examined three castrated male and three spayed female dogs with an equal number of controls of the appropriate sexes. He operated on them at the age of 3 years and a weight of 8-9 kg. and killed them after 1 month. He found no changes in the adrenals. No experiments on cats have been found. (6) Other species. Marrassini and Luciani [1911] recorded the adrenal and body weights on five spayed sheep with four controls, but the age at operation was not given and the age at death was 5-8 years in the controls and 1 year in the castrated animals, so-that no conclusions can be drawn. Of nineteen castrated male cattle, eleven were of an age comparable to that of the five normal animals, namely in the range of 3-9 years, and no clear-cut differences in the adrenal weights were observed. In addition to mammals, experiments have been reported in frogs and chicks. Ciaccio reported hypertrophy of the adrenal to double the normal size in frogs killed 15 days after gonadectomy, the increase being entirely cortical. Soli, in fifteen capons, killed 2-6 months after castration, with fifteen control cocks of the same age, found slight decrease in the volume of the adrenals. Marrassini and Luciani recorded the adrenal weights in nineteen capons and six cocks of widely varying ages and post-operative intervals and no constant change is observed in this series. (2) Technique. Preliminary experiments were carried out several years ago on young rats which were spayed or castrated between the ages of 50 and 85 days and killed after intervals varying from 1 to 3 months (Table I, Exp. I; Table II, Exp. IX). In these animals the adrenals of the spayed females were markedly atrophied and those of the castrated males were somewhat hypertrophied. Since the change in the latter was quite variable, the experiment was repeated with greater care on a larger series of male animals (Table I, Exp. II). These rats were castrated at the age of 100 days and

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11 EFFECT OF GONADECTOMY ON GLANDS. killed 50 days later, and under these conditions the adrenals were not hypertrophied. The difference in these findings might be due either to the difference in age at operation or in the post-operative interval. The subsequent experiments were therefore planned with the idea of obtaining accurate information as to the immediate and late effects of gonadectomy on immature and mature rats of both sexes. The rats were of the same strain as those used in our previous experiments. They were bred in the laboratory in litters limited to six at the age of 1 day. Those weighing less than 30 g. at weaning at 28 days were discarded. The majority weighed g. at 28 days. As far as possible the rats of each litter were paired by sex and weight, and one of each pair was used for operation, the other as the control in the same experiment. The diet was that used in our previous experiments [Andersen, 1932 a] and was a McCollum B diet with the addition of 5 p.c. of dried yeast and a ration of fresh lettuce twice a week. Previous experiments have shown that healthy male rats of our colony mature before the seventieth day [Andersen, 1932 b]. The immature animals were gonadectomized at 21 or 22 days and the mature ones at days. In a few groups the mature animals were several weeks older than this. In addition to the normal controls there were also vasectomized controls for the males. The vasectomy was performed by a double ligation with excision of 3-5 mm. of the vas between the ligatures and was always bilateral. In castration the entire testes and epididymes were removed; in the males castrated after maturity the epididymes were examined for the presence of mature motile sperm, which were found in every case. The peritoneal route was used in the operations on the 21-day-old male rats, while in the 80-day-old males the operation was performed through the scrotum. Daily vaginal smears were made on all the older female rats and only those having regular cycles were used. Rats operated on at 80 days were not previously smeared. The ovariectomy was done by the dorsal route, through small bilateral incisions in the paravertebral region. The ovary, tube, adjacent fat, and a small portion of the uterus were removed, and the remaining vessels and the cut end of the uterus were included in a single ligature. The operations were performed under ether anesthesia and with aseptic technique. At autopsy the operative wound was examined for infection and this was present only in several of the mature vasectomized and castrated animals. These rats were discarded from the series and replaced by uninfected ones. In one series of mature females, half of the animals were spayed during oestrus and half during dicestrus. The animals of this group were somewhat older than the others, the age varying from 100 to 200 days 11

12 12 D. H. ANDERSEN AND H. S. KENNEDY. at the time of operation. Most of the experiments were performed during the late spring and early summer. The animals gonadectomized at 21 days were sacrificed 1, 3, or 9 weeks after operation, and the mature groups were sacrificed 1, 3, 6, or 8 weeks after operation. The animals were killed with chloroform, and the thyroid, adrenal, and pituitary were quickly dissected out, placed in a closed weighing bottle, and weighed to 01 mg. These organs were then fixed in Zenker's fluid or formalin. Fresh smears were obtained from both epididymes of all vasectomized and control males. These were suspended in saline and studied for the number, motility, and maturity of the sperm. The actual weight of the organs in question and the relative weight in terms of mg./kg. of body weight were obtained for each animal and the mean of the actual and also of the relative weights of each organ were calculated for each group. The probable error of the mean was calculated by the formula used in our previous experiments [Andersen and Kennedy, 1932]. The following organs were sectioned and stained with hsematoxylin and eosin: in the castrated males, the thyroid and adrenal; in the vasectomized males, the testes; in control males, the thyroid, adrenal, and testes; in the spayed and control females, the thyroid and adrenal. The adrenals were sectioned in three males vasectomized at 80 days and killed after 1 week. In the thyroid and testes a few sections were obtained through the thickest portion of the organ. In some animals of each group the adrenals were fixed in Zenker's and serially sectioned; every tenth section was mounted and then stained with ha3matoxylin and eosin. In some instances, as reported below, tracings were made of these sections and the volume of the cortex and medulla measured with a planimeter. In other animals of each group, the adrenals were fixed in formalin, and frozen sections were made and stained for fat with Scharlach R. The pituitaries are yet to be studied histologically and will be reported later. (3) The adrenal weight after gonadectomy. (1) Male rats castrated and vasectomized before maturity. In all, fortysix rats were used in this portion of the experiment. Four of these were controls killed at the age of days to obtain the endocrine weights at the time of operation. Six castrated and six normal animals were killed at the age of 28 days (Table I, Exp. III). Four each of castrated, vasectomized, and control animals were killed at the age of 42 days (Table I, Exp. IV). No significant differences were found in the adrenal weights of the castrated animals as compared with their controls in either

13 EFFECT OF GONADECTOMY ON GLANDS. 13 of these experiments. Neither control nor vasectomized animals had mature motile sperm in the epididymes at 28 or 42 days of age. There were mature motile sperm in the epididymes in all control and vasectomized animals at 85 days. The castrated animals had significantly larger adrenals than the control animals, while the adrenals of vasectomized rats were similar to those of the controls (Table I, Exp. V). The figures for the relative weight of the adrenals of normal animals show a sharp decline \ -v-..-. Castrated 780 \ ---O Vasectomizel 260 Control MX 180 N 0f 160 \ Adrenals body 140 weight Thyroid w..., 40 - i} Pituitary Age in days Text-fig. 1. The relative weights of the pituitary, thyroid, and adrenals after castration or vasectomy at the age of 21 days-male rats. with age, while in castrated animals there is a marked lag in this decrease (Text-fig. 1). These figures may be supplemented by the data of Hatai [1915]: in rats castrated at days and killed after days he found weights similar to those reported here, showing that even after this long interval the glands do not come down to the size of those of normal animals. (2) Male rats castrated and vasectomized after maturity. Forty-three rats, some of which were probably not mature, were used in the preliminary experiments, and forty-eight were used in the more carefully

14 14 D. H. AiNDERSEN AND H. S. KENNEDY. standardized experiments. The latter were castrated at days and killed after 1, 3, and 8 weeks. In all cases the epididymes of the vasectomized and control animals contained mature motile sperm and the weights of the testes were similar, showing that 8 weeks is not long enough after vasectomy for any significant atrophy of the testis to take place. The organ weights of these two groups were parallel in each case X. Castrated. Vasectomized 2.00 * - Contro) 180- ik5 of 160 bcod 140 weig't 12.0 ) Adreia den to0 " '~*w -..,I Thyroid iej;;;;; qj Pituitary Interval after spaying in weeks Text-fig. 2. The mean relativeweights of the pituitary, thyroid, and adrenals after castration at 80 days of age-male rats. One week after castration the adrenals were slightly enlarged (Table I, Exp. VI). A much longer series would be necessary to ascertain whether this enlargement is significant, but it does not appear so in the present series. After 3 and 8 weeks the relative adrenal weights were similar to those of the controls (Text-fig. 2). The adrenal hypertrophy after castration found in the first preliminary experiment may be attributed to the probable immaturity of some of the animals. (3) Female rats spayed' before maturity. This series included twelve operated and ten control animals (Table II, Exps. X, XI). Four of the controls were killed at the age of days to show the conditions at the time of operation. In the animals which were spayed at 21 days and killed

15 EFFECT OF GONADECTOMY ON GLANDS. 15 after 1 week no adrenal changes were found as compared with the controls. Since the female rats of our colony are mature after 50 days, at 85 days they all show cyclic changes in the adrenals. The figures obtained from our previous work on adrenal changes associated with cestrus are therefore utilized as control data for the spayed animals killed at 80 days [Andersen and Kennedy, 1932]. These control rats were young mature animals not classified accurately as to body weight, but all had regular cycles (Table II). 300 h0 th Adrenals 2.40 ' ' s zoo *.. Control x Spayed > Oestrus control 10 \ D Dioestrus control of hoay 140. weiglt 12.0 so Operation Ix.AD) Pituitary c Age in days Text-fig. 3. The relative weights of the pituitary, thyroid, and adrenals after spaying at the age of 21 days-female rats. In the spayed rats killed at 85 days, the vagina was found open and the uterus was immature in each case. No gross ovarian tissue was found. The mean adrenal weight of these rats is about the same as that in the controls in dicestrus (Table II, Exp. XI; Text-fig. 3). (4) Female rats spayed after maturity. Twenty-one rats were used in the preliminary experiment, of which twelve were spayed and eleven were controls (Table II, Exp. IX). They were spayed between the ages of 53 and 73 days and killed after 1-2 months. Thirty-six spayed animals were included in the later series. The control data was obtained from our previous work on cestrus changes [Andersen and Kennedy, 1932]. The

16 16 D. H. ANDERSEN AND H. S. KENNEDY. operation was performed between the ages of 80 and 200 days, and the animals were killed after intervals of 1, 3, 6, and 8 weeks. Seventeen animals were killed 1 week after spaying. The adrenal was hypertrophied with a mean relative weight similar to that found at cestrus and well above that found at dicestrus. Seven of the animals were spayed at cestrus and six at dicestrus, while in four the phase of the cycle was not noted. No difference was found in the mean adrenal weight of these three groups (Table II, Exp. XII; Text-fig. 4). X-..*Spayed Oestrw control 280 D - Dioestrus control 240~~~~~~0 220 Adrenals zoo. *s \. 180 of body 14 wei ght ) 100 AD.. 80X, A Thyroid 6040 XA:.I Pititary 40 t Interval afer spaying in weeks Text-fig. 4. The mean relative weights of the pituitary, thyroid, and adrenals after spaying at the age of days of age-female rats. The animals killed 3 weeks after operation were in two groups, of four and five rats respectively (Table II, Exp. XIII). Group A was killed in the autumn and Group B was killed in the early summer. The adrenals in Group B were atrophied to a degree similar to that found in animals killed after a longer interval. It seems probable that some factor, probably seasonal, delayed the progress of the usual changes. In the rats killed 6 and 8 weeks after operation the relative adrenal weight was about 30 p.c. below that found at cestrus and only slightly

17 EFFECT OF GONADECTOMY ON GLANDS. above that found in young mature males (Table II, Exps. XIV, XV). These figures corroborate those obtained in the preliminary experiment (Table II, Exp. IX). (4) Histological observations. Serial sections were made of both adrenals of two or three of the castrated male rats and of an equal number of controls of Exps. III, V, VI, VII, and VIII (Table I). Serial sections were also made of the adrenals of three vasectomized rats of Exp. VI. Frozen sections stained with Scharlach R were made of the adrenals of two or more castrated rats and an equal number of controls from each of these groups. No differences were found between the adrenals of the operated and control animals of Group III, in which the rats were castrated at 21 days and killed at 28 days. In Group V, however, in which the rats were castrated at 21 days and killed at 85 days, the adrenals of the operated animals retained to some extent the structure found in immature animals (P1. I). The cortex was wider than that of the controls, and much less clearly divided into zones. It was similar in width to that of the normal dicestrus female [Andersen and Kennedy, 1932]. The zona glomerulosa was narrow and less clearly demarcated than in the normal male. The lipoid had approximately the same distribution, but was a little less abundant. In the rats castrated at 80 days and killed after 1 week there was a slight increase of lipoid in the fasciculata and marked engorgement in the inner third of the cortex (P1. I). This was not found in the vasectomized animals. No difference was observed between the adrenals of the animals of Groups VII and VIII, killed 3 and 8 weeks after castration, and their controls. In the study of the female spayed rats, serial sections were made of both adrenals of at least two control and two operated rats in each of Exps. X, XI, XII, and XIV (Table II), and fat stains were done on the adrenals of at least two rats of Exps. XI, XII, and XIV. The animals used in our previous studies on the cestrous cycle [Andersen and Kennedy, 1932; Andersen, 1933] served as controls for the mature animals, while controls of the same age were studied in the case of immature animals. The adrenals of animals killed at the age of 28 days were all similar regardless of sex or gonadectomy. In the female rats spayed at 21 days and killed at 85 days of age the fasciculata contained a moderate amount of lipoid, but not as much as is present in animals at cestrus. The groups of cells near the medulla were not prominent and the glomerular zone was sharply demarcated in some cases and not in others. The glands resembled PH. LXXIX. 2 17

18 18 D. H. ANDERSEN AND H. S. KENNEDY. those of the castrated males in the corresponding experiments. In the animals spayed at 80 days and killed after 1 week, the adrenals were indistinguishable from those found at cestrus in normal animals (P1. II). This was true regardless of whether the operation was performed at cestrus or dicestrus. The engorgement of the inner third of the cortex was marked. In those of Group XIV, killed 6 weeks after spaying, the cortex was greatly narrowed, with a sharply demarcated glomerulosa, a moderate amount of lipoid in the outer half of the fasciculata. The more striking changes are those in the inner half of the cortex, where the capillaries show extreme congestion with narrowing of the cell columns, decrease in size of the cells, pyknosis of most of the nuclei, and a deeper eosinophilic stain in the cytoplasm (P1. II). In summary, we may say that the adrenals of animals spayed before maturity resemble those of their controls of the same age until the latter have become mature. The adrenals of the operated animals then retain in part the structure of those of the immature animals. In the case of gonadectomy after maturity there is a temporary increase in lipoid in the fasciculata, more marked in the females, with congestion in the inner third of the cortex. In the male 3 weeks after castration the adrenals appear similar to those in normal animals. The process of regression is slower in the female, with degenerative changes in the inner portion of the cortex and marked congestion of the capillaries. (5) The volume of the cortex and medulla. The relative volumes of the cortex and medulla of two male castrated rats and their normal controls from Exp. III and an equal number from Exp. IV have been measured and calculated by the same method used in our previous work [Andersen and Kennedy, 1932]. Similar studies have been made on two female rats of Exp. XII. This was done by making tracings of every tenth section (cut at 5,u), measuring the area of the adrenal and of the medulla with a planimeter and calculating the percentage of the total volume occupied by the medulla. On the assumption that the specific gravity of the cortex and medulla is approximately the same, the relative weight of each was calculated. The results are given in Table III. The series is too small to give more than an indication of the volume changes. It is obvious, however, that the medulla is about equal in size in the four adult males of the series, and slightly larger in the two females in the series, as well as in the female controls. This sex difference may only be apparent as the greater lipoid content of the female cortex would make for greater shrinkage in the process of

19 EFFECT OF GONADECTOMY ON GLANDS. preparing the sections, and the medulla would therefore appear slightly larger. The change in size in the gonadectomized animals is mostly if not entirely due to change in size of the cortex. TABLE III. The weight of the adrenal cortex and medulla of the rat. Adrenals Rat A - Weight Cortex Medulla Experiment No. in g. mg. mg./kg. mg./kg. mg./kg. Males castrated at 21 days, killed 1149 Op at 85 days 1147 Op ' Male controls aged 85 days 1151 C C * Females spayed at 80 days, killed 1413 Op at 87 days 1415 Op P8 Female controls aged days: CEstrus Av. of Dicestrus Av. of Data on female controls from Andersen and Kennedy [1932]. II. THE THYROID. (1) Review of literature. So far as could be ascertained the earliest observations as to the effect of castration on the thyroid are those of Cecca [1904] and of Tescione [1904], who concluded that the operation was followed by a temporary hypertrophy and later by atrophy. Martini [1904] reported changes which he interpreted as those of hypersecretion. These papers are not available to us, and the species investigated are therefore not known. Most of the subsequent work has been done on rodents. (1) Rabbit. Soli [1909] reported hypertrophy of the thyroid after gonadectomy followed by decrease in the size of the gland after about 3 months in seven male and three female gonadectomized rabbits, as compared with an equal number of controls of the same age and sex. Engelhorn [1912], in a paper which we have been unable to obtain, reported hyperplasia with increase of colloid and flattening of the epithelium in the thyroids of female rabbits 3 months after spaying. Bell [1913] recorded similar findings in castrated rabbits and interpreted them as evidence of increased thyroid activity. Livingston [1916], in a long series of rabbits which, however, showed extreme variations in age, weight, and post-operative interval, concluded that the thyroid atrophied slightly in the male, but showed no change in weight after gonadectomy in the female. He gave a detailed report of his experiments, and inspection of the thyroid weights reveals such wide variations as to make the

20 20 D. H. ANDERSEN AND H. S. KENNEDY. difference between the mean weights of the castrated and control animals insignificant. Okuneff [1922] in seventeen rabbits killed 2-5 months after castration found an increase in colloid in the follicles with a decrease of lipoid in the epithelial cells, which he interpreted as evidence of increased thyroid secretion. Saito [1930], giving no details of his experiments, reported decrease of thyroid weight in both sexes with an increase in iodine content. In the rabbit, therefore, no adequately controlled studies have been reported in sufficient detail to establish the presence or direction of a change in weight of the thyroid after gonadectomy. EngelIhorn, Bell, and Okuneff are unanimous in stating that there is an increase in colloid in the follicles, and the two former workers agree that the epithelium is flattened. (2) Guinea-pig. No observations on the weight of the guinea-pig thyroid after castration have been found. Constantini [1929] found no constant change in the histological picture 1 month after castration. Chouke [1930] confirmed this and also reported no change in the number of mitoses in fourteen female guinea-pigs, killed at intervals of days after operation, as compared with six controls, none of which was in heat. There was an increase in the three controls killed during heat. (3) Rat. The first and most careful observations on weight changes in the rat thyroid are those of Hatai [1915]. The plan of the experiments has been given in the above discussion of the observations on the adrenals. The male rats castrated before the twenty-second day and killed 3-5 months later had marked thyroid atrophy, but in the females spayed before maturity the mean thyroid weight showed an insignificant increase in weight. In the male rats castrated after maturity there was a slight thyroid atrophy. The females spayed after maturity varied in their response: those spayed at days and killed after days had marked atrophy, while those castrated at days and killed after days showed a slight increase in the weight of the thyroid. Inspection of the figures shows that this discrepancy is due to a change in the controls. The mean thyroid weight is approximately the same for the two groups spayed after maturity, but the weight of the thyroids in the older control group is very much less than in the younger control group and is comparable to that found in our older ancestrous animals (data unpublished). Matsuyama [1921] gave no weights, but observed histological changes in rats killed several months after castration. These consisted of flattening of the epithelium with an increase of colloid in the follicles, which stained more deeply with eosin. Furuy a's observations [1924] on

21 EFFECT OF GONADECTOMY ON GLANDS. the altered compensatory hypertrophy in spayed rats after removal of one lobe of the thyroid are based on few animals and also on the false assumption that the two lobes of the thyroid are of equal weight. It can be disregarded. Minowada [1928] with twelve castrated and five control male rats observed a decrease in the size of the follicles in the former. His figures show a slight decrease in the mean adrenal weight of the gland. Bokelmann and Scheringer [1932] found some atrophy in eleven adult females killed 5 weeks after operation as compared with an equal number of controls, and obtained similar results in a somewhat larger number of animals killed 6 months after operation. Calculations based on their figures show that the difference between the mean and control thyroid weights is not significant because of the wide variations. They analysed the glands for iodine and reported an increase, which also does not bear statistical analysis, but which confirms the findings of Saito in rabbits. We are left with the impression that castration produces atrophy of the thyroid in the rat, but the variation of the weights in the protocols of Bokelmann and Scheringer, and the hypertrophy reported by Hatai in the females spayed before maturity and those spayed after the age of 6 months leaves room for doubt. The histological report of Mats uyama does not regard the differences of age, sex, and post-operative interval which, according to Hatai, are so significant. (4) Other species. Bell [1913] found no change in the thyroid of the cat after gonadectomy. No investigations on dogs have been found. Merschejewsky in Korentschewsky's laboratory is reported by the latter [1914] to have found atrophy of the thyroid in castrated oxen and horses. Okuneff [1922] observed an increase of colloid in the follicles, and a decrease of lipoid in the epithelial cells of the thyroid of oxen. No constant change was found by Soli [1909] in capons. Atrophy of the thyroids was reported in eunuchs by Tandler and Gross [1913]. In all these experiments it is obvious that only Hatai investigated the effect of castration in relation to the age at operation, and only Chouke has noted whether the female controls were killed during cestrus, or whether their reproductive activity was normal. (2) The thyroid weight. The data were obtained from the animals used for the observations on the adrenals, except in the case of mature female controls, for which the previous report on cestrous changes was utilized [Andersen, 1933]. In all cases the vasectomized rats resembled the controls.., 21

22 22 D. H. ANDERSEN AND H. S. KENNEDY. (1) Male rats castrated before maturity. There was no significant difference between the mean relative thyroid weights of the castrated and control rats killed at 28 and 42 days. At 85 days there was a slight but definite decrease in the thyroid weight of the castrated rats (Text-fig. 1). (2) Male rats castrated after maturity. No change in thyroid weight was observed after 1 week. After 3 and 8 weeks a moderate atrophy was noted, which was not statistically significant (Text-fig. 2). (3) Female rats spayed before maturity. No change was found at 28 days. However, at 85 days the mean adrenal weight of the thyroid was definitely below normal (Text-fig. 3). (4) Female rats spayed after maturity. The thyroid showed some atrophy in each series. This was significant after 1 week, and after 3 weeks was greater in Group B than in Group A. After 8 weeks the mean relative thyroid weight was about 25 p.c. below that found in normal animals (Table II, Exp. XI; Text-fig. 4). (3) Histological observations on the thyroid. Sections were made of the thyroids from four male rats castrated at the age of 21 days and killed at the age of 28 days; from four male rats castrated at the age of 21 days and killed at the age of 85 days; and from an equal number of male controls for each group. No difference was found at 28 days between the thyroids of the operated and those of the control animals. However, in those killed at 85 days a distinct difference was noted. In all rats the peripheral follicles are larger than the central ones and have more colloid and a lower epithelium. In the castrated animals the thyroid is definitely smaller, and the irregularity of the decrease in weight is at least partly explained by the presence of various amounts of fat and muscle removed and weighed with the glands in almost every instance. In the castrated rats the follicles are on the whole smaller than the follicles in corresponding portions (i.e. periphery or centre) of the control glands. In the central portion the epithelium is lower, with a darker, less abundant cytoplasm and more distinct nuclei which lie in about the centre of the cells. The epithelial border is more sharply demarcated. The colloid appears denser, is more eosinophilic, contains fewer vacuoles and includes fewer desquamated cells (Text-fig. 6). These changes are found to a lesser degree in the peripheral vacuoles. In the controls many of the thyroid follicles contain hyperchromatic nuclei, apparently originating from desquamated epithelial cells. Some of the nuclei of the epithelial cells are also small and hyperchromatic. The colloid, by comparison with the operated animals, is pale, even in consistence, and contains an abundance

23 EFFECT OF GONADECTOMY ON GLANDS. of vacuoles. The blood vessels and capillaries contain more blood and are therefore more conspicuous. In thyroids of four male rats castrated at 80 days and killed after I week, a suggestion of similar changes is found as compared with the four controls of the same age. These changes are not sufficiently striking so that the diagnosis of castrated as against control animals can be made from inspection of the thyroid. The most definite change is in the colloid, which appears denser, more eosinophilic, and has a much less foamy appearance. In the four males castrated at 80 days and killed after 8 weeks the thyroid changes, as compared with the controls of the same age, are the most marked of those found in any group. They resemble those found in the animals castrated at 21 days and killed at 85 days. The decrease in the size of the follicles is obvious. The epithelium is flattened so that the nucleus occupies most of the cell volume and there appears to be only cell membrane between the nucleus and the lumen of the follicle. In the central follicles the nucleus remains round, but in the peripheral follicles it is flattened and hyperchromatic, and in some places the cells are several times as wide as they are thick in the radial axis of the follicle. Blood vessels are less conspicuous than in the normal thyroids. We may therefore say that there is a definite change in the histological picture of the male thyroid after castration, whether the operation is performed before or after maturity, and that this change is clear-cut in rats killed 8 weeks after castration. The observations on the female thyroid after spaying are based on sections of both lobes from four animals which were spayed at the age of 80 days and killed after 6 weeks, at which time the thyroid shows a definite decrease in weight. For a control, sections were made of both lobes from nine normal rats which had had regular cycles of 5 days or less; four of these were killed at cestrus and five at dicestrus. In eight of these rats the histological picture was similar, and resembled that found in the normal males of the same age. The impression was that on the whole the colloid was a little more vacuolated and the cells a little larger and more clearly outlined than in the males, but it was impossible to make an accurate diagnosis of male thyroid as against female thyroid by inspection of the sections. The ninth thyroid, that of a rat at dicestrus, somewhat resembled those of the spayed animals. The thyroids of the spayed animals were similar to those found in the male rats castrated at 80 days and killed after 8 weeks. It was therefore possible to recognize a thyroid from inspection of the sections as that of a normal or gonadectomized animal, but it was not possible to state the sex in either case. 23

24 24 D. H. ANDERSEN AND H. S. KENNEDY. III. THE PITUITARY. (1) Literature. The literature on the effect of castration on the pituitary has been recently reviewed by Schenk [1927] and by Severinghaus, Engle and Smith [1932]. Most authors agree that there is hypertrophy, but the histological changes are still a matter of dispute. As we have as yet made no adequate histological studies on the pituitary, the literature will not be reviewed here. (2) The weight of the pituitary. The data were obtained from the animals used for the adrenal studies, except for the mature female controls, which have been previously reported [Andersen, 1933]. In all cases the vasectomized rats resembled the controls. (1) Male rats castrated before maturity. At 28 days the mean relative weight of the pituitary in the castrates was greater than that of the controls, but the probable error was sufficient so that the difference was not significant. At 42 days, however, the pituitary was definitely larger. Calculation shows the relative weight of the pituitary to be the same in 21-day controls, and in rats castrated at 21 days and killed at 28 or 42 days, while in the normal animals the relative weight of the pituitary decreases with age. In the castrated animals killed at 85 days the relative weight of the pituitary is less than in the 42-day-old castrated rats, but is about 42 p.c. above that of the controls (Table I; Text-fig. 1). (2) Male rats castrated after maturity. The relative weight of the pituitary does not increase much during the first week after castration, but is about 26 p.c. greater than in the controls after 3 weeks. By the eighth week it is about 57 p.c. greater. The pituitary, therefore, hypertrophies gradually after castration throughout the 8-week period studied (Table I; Text-fig. 2). (3) Female rats spayed before maturity. No change was found in rats killed at 28 days. At 85 days the mean relative pituitary weight of the spayed animals lies between that of the controls in oestrus and of those in dicestrus (Table II, Exp. XI; Text-fig. 3). (4) Female rats spayed after maturity. After 1 week the pituitary was significantly larger in the rats which were spayed during cestrus than in those spayed during dicestrus (Table II, Exp. XII). In the two groups of rats killed after 3 weeks the mean relative pituitary weight was greater in Group B, which also showed more advanced adrenal and thyroid

25 EFFECT OF GONADECTOMY ON GLANDS. changes, but lay between the mean relative pituitary weight in normal rats at cestrus and dicestrus in both groups. After 8 weeks the mean relative pituitary weight had increased to but not above the normal cestrous weight. There is, therefore, no hypertrophy of the pituitary after spaying in the mature female rat (Table II; Text-fig. 4). IV. HISTOLOGICAL OBSERVATIONS ON THE TESTES OF VASECTOMIZED ANIMALS. Sections have been made through the thickest portions of one testis from each of four vasectomized and four normal control rats of each group in which vasectomized controls were used. In no case do the testes of vasectomized rats differ from those of the controls in the histological appearance of either tubules or interstitial cells. Vasectomy, whether performed before or after maturity, therefore, does not affect spermatogenesis in the period of eight weeks. No abnormalities were found in the approximate quantity, appearance, or motility of sperm obtained from fresh smears of the epididymis. V. DISCUSSION. It is obvious from inspection of the data in Tables I and II that the effect of castration on the adrenals in the rat varies not only with the sex and with the age at operation, as pointed out by Hatai [1915], but with the interval after operation. These experiments have been planned in consideration of these points, and with the advantage of knowledge of the cyclic changes in the female adrenal. As in the case of cestrous hypertrophy, changes have been found only in the cortex. In normal rats 21 days of age, the relative adrenal weight is the same in both sexes. At 85 days the relative adrenal weight is more than twice as great in the normal female rat as in the normal male. In animals of both sexes gonadectomized at 21 days and killed at 85 days, the relative adrenal weights lie between those of the male and female controls. They are somewhat greater in the spayed female than in the castrated male. It is curious that removal of the gonads in immature animals, at an age when the adrenals show no sex differences, should not permanently remove sex differences in that organ. It is useless at present to speculate as to whether this is due to a primary sex difference in some other organ, perhaps the hypophysis, or whether the presence of the gonads during the first 3 weeks of life has permanently affected the endocrine glands, even though these 25

26 26 D. H. ANDERSEN AND H. S. KENNEDY. organs were not functioning in a reproductive capacity, and had not at that age produced any discernible sex differences in the pituitary or adrenal. A similar problem confronts us in regard to the effects of the operation on the mature animals. In these mature rats, however, another interesting question is brought up by the temporary increase in cortical lipoid, more marked in the females. The effect of an operation per se has been ruled out in rats killed 1 week after operation because of the absence of this change after vasectomy. This hypertrophy is followed by return to normal in the male and by degenerative changes in the female. By 6 weeks, however, the female adrenals have reached their final low weight in our series. The changes in the spayed female may be partly due to the absence of cestrus. The fact that the decrease in weight is greater in spayed animals than in animals in dicestrus may be interpreted either as meaning that there is an adrenal-stimulating hormone present during dicestrus as well as cestrus, or that the regressive changes in the adrenal after cestrus are not completed before the arrival of the next cycle in the rat. If the latter were true one would expect the adrenals to be the same in spayed animals and in animals in dicestrus of species having long periods between cestrus. This would explain the negative findings of some workers in the dog, rabbit, and guinea-pig. The confusion in the literature in regard to adrenal changes after castration may be explained by the usual disregard of the presence of infection, and the frequent disregard of other factors such as age and weight at operation and autopsy, the presence or absence of cestrus in the female controls, the uniformity of diet, breed, and post-operative interval. The question may be considered unsolved in the rabbit and dog. There is probably atransitory hypertrophy in mature male guinea-pigs, with a normal adrenal weight in both sexes some weeks after operation. The effect in the rat and mouse has been shown to vary with the sex of the animal and the age at operation. The presence of the x zone in male mice castrated before maturity is comparable to the finding of adrenals resembling those of immature animals in our similar experiments on rats. Gonadectomy produces atrophy of the thyroid of the rat in both sexes and regardless of the age of operation. It is not definitely established after 3 weeks, but is clear after 8 weeks. It is most marked in the mature female 8 weeks after spaying. The impression obtained on reviewing the literature on the subject is that gonadectomy produces thyroid atrophy in most of the species investigated, although much of the work is open to criticism.

27 EFFECT OF GONADECTOMY ON GLANDS. The hypertrophy of the pituitary after gonadectomy is well known, and the only contribution to the problem which is presented here consists of specific data on the sex, age, and time intervals involved. In rats of both sexes gonadectomized at 21 days and killed at 85 days, the pituitary weighs approxrimately mg. per kg., which is greater than that of the male controls and about the same as the female controls at dicestrus. In the mature male rat the pituitary hypertrophy is gradual and continues throughout the 8-week interval studied. In the female there is but little change in the weight of the pituitary in 1 week. Those spayed during cestrus have heavier glands than those spayed during dicestrus, but both are within the weight range found at dicestrus. At 3 and 6 weeks the gland is slightly larger than at dicestrus, while after 8 weeks it has regained the cestrous weight. Therefore there is no increase in the pituitary weight of the spayed female over the normal range of weight. At 8 weeks after operation the pituitary is larger in the female than in the male of the corresponding experiment. It is interesting to observe that the changes in the thyroid, adrenal, and pituitary of gonadectomized animals as compared with controls, are not established until the controls are mature, in the case of animals gonadectomized at 21 days; and they are not established at 3 weeks and are established at 8 weeks after operation in the case of the animals gonadectomized after maturity. The various changes, therefore, require approximately the same time and are undoubtedly related. The interpretation of the significance of these changes in the endocrine glands after castration cannot yet be made. Recent studies on the relation of reproductive changes to metabolism such as those of Okey and Boyden [1927] on the cyclic changes in blood lipoids in women and those of Kaufmann and Miihlbock [1931] on changes in the lipoid metabolism after ovariectomy in women indicate a probable source of enlightenment on the question. The normal number, appearance, and motility of spermatozoa up to 8 weeks after vasectomy, together with the normal weights of the endocrine glands and normal histological appearance in these animals is an indication that the operation in itself has no effect and that vasectomy is in no way comparable to castration in a physiological sense, at least for the first 8 weeks. The fact that the vagina opened before the age of 85 days in all of our animals spayed at 21 days is proof that the opening of the vagina is not dependent entirely on coincident ovarian activity. The uterus was immature and no ovarian tissue was found in these animals. 27

28 28 D. H. ANDERSEN AND H. S. KENNEDY. VI. CONCLUSIONS. I. The effect of gonadectomy on the adrenals of the rat varies with the sex of the animal, the interval after operation and the age at operation. Measurements of the volume of the cortex and medulla in a small group of rats show that effects of castration are evident only in the cortex, the medulla remaining approximately uniform in volume and showing no histological changes. The findings in the various experiments are as follows: (1) In male rats castrated at the age of 21 days and killed 1 or 3 weeks after operation, the adrenal shows no changes in weight or histological appearance as compared with the controls. Nine weeks after operation the adrenals of the castrated animals are hypertrophied, contain more lipoid than the controls, and histologically resemble the adrenals of immature animals. (2) In female rats spayed at the age of 21 days and killed after 1 week the adrenals resemble those of the controls of the same age. When killed after 9 weeks the adrenals have approximately the same relative weight as those of controls in dicestrus and weigh more than those of the males of the corresponding experiment, but resemble them histologically. (3) In male rats castrated after maturity and killed after 7, 21, or 56 days there is no significant change in the adrenal weight. One week after operation there are histological changes consisting of increased lipoid in the fasciculata and congestion of the reticularis. The histological appearance returns to normal. (4) In female rats spayed after maturity and killed after 1 week the adrenal is hypertrophied and has the histological appearance of the adrenal at cestrus. This is true whether the rat was in cestrus or in dicestrus at the time of operation. In one group of rats killed after 3 weeks the adrenals showed marked atrophy, while in a second group they did not. The adrenals of rats killed 6 or 8 weeks after spaying are all atrophied, with decrease of lipoid in the fasciculata and degenerative changes and engorgement in the reticularis. II. No changes are found in the thyroid 1 week after castration, but there is atrophy after 3 weeks in some experiments. A definite atrophy is apparent 8 weeks after castration in all cases, which is accompanied by characteristic histological changes. III. There is hypertrophy of the pituitary after castration of either immature or mature male rats, which is progressive throughout the 8-week interval studied. In the spayed female rats the pituitary weight does not

29 EFFECT OF GONADECTOMY ON GLANDS. 29 become greater than that found at cestrus, but is somewhat greater than in the castrated male rats. IV. A critical survey has been made of the literature on the effect of gonadectomy on the adrenal in various species. No constant change has been definitely proven in the rabbit, dog, ox, sheep, and capon. There is a temporary hypertrophy after spaying in the adult female guinea-pig, and a hypertrophy was found 2 weeks after gonadectomy in the frog. Changes in the rat and mouse vary with the sex and age at operation. The literature on the thyroid is contradictory, but the balance of evidence is for atrophy after gonadectomy. V. Vasectomy does not affect the number, appearance, or motility of the spermatozoa, nor does it affect the pituitary, thyroid, or adrenal within a period of 8 weeks after operation. VI. Spaying before maturity delays, but does not prevent the opening of the vagina. REFERENCES. Altenburger, H. (1924). Pftfigerm Arch. 202, 668. Andersen, D. H. (1932 a). J. Physiol. 74, 49. Andersen, D. H. (1932 b). Ibid. 74, 207. Andersen, D. H. and Kennedy, H. S. (1932). Ibid. 76, 247. Andersen, D. H. (1933). Proc. Soc. exp. Biol., N.Y. 30, 657. Bell, B. (1913). Lancet, 809 and 937. Bokelmann, 0. and Scheringer, W. (1932). Arch. Gyniuk. 148, 1. Cecca (1904). Boll. della 8cienza med. Chouke, K. S. (1930). Endocrinology, 14, 12. Ciaccio, C. (1903). Anat. Anz. 23, 95. Constantini, P. (1929). Ramsegna internaz. clin. terap. 10, 718. Deanesly, R. (1928). Proc. Roy. Soc. B, 103, 523. Dick, G. F. and Curtis, A. H. (1912). Surg. Gynec. Ob8tet. 15, 588. Engelhorn (1912). Schilddruese und weibliche GeAchlecht8organe. Habilitationsschrift, Erlangen. Feodosyeff, N. E. (1905). Rus8k. Vrack. 5, 135. Furuya, K. (1924). Biochem. Z. 147, 425. Hatai, S. (1915). J. exp. Zool. 18, 1. Howard-Miller, E. (1927). Amer. J. Anat. 40, 251. Kaufmann, C. and Miuhlbock, 0. (1931). Klin. Wschr. 10, 696. Kolde, W. (1913). Arch. Gynak. 99, 272. Korentschewsky, W. G. (1914). Z. exp. Path. 16, 68. Leinati, F. (1929). Atti Acc. Fisiocr. Siena, 3, 385. Livingston, A. E. (1916). Amer. J. Physiol. 40, 153. Marrassini, A. (1906). Sperimentale, 60, 197. Marrassini, A. and Luciani, L. (1911). Arch. ital. Biol. 56, 395. Martini (1904). Accad. di Med. Torino. Masui, K. and Tamura, Y. (1926). J. College Agric., Imp. Univ. Tokyo, 7, 353.

30 30 D. H. ANDERSEN AND H. S. KENNEDY. Matsuyama, R. (1921). Frank. Z. Path. 25, 436. Minowada, M. (1928). Acta dermat. 12, 668. Okey, R. and Boyden, R. E. (1927). J. biol. Chem. 72, 261. Okuneff, N. (1922). Zbl. allg. Path. path. Anat. 32, 531. Parhon, C. I., Marza, V. and Kahane, M. (1926). C. B. Soc. Biol., Paris, 95, 781 and 795. Pianese, F. (1929). Arch. Ostet. Ginec. 16, 529. Raineri, G. (1908). Ibid. 30, 744. Rigano-Irrera, D. (1929). Boll. Soc. ital. Biol. sper. 4, 973. Saito, T. (1930). Fol. endocrin. Jap. 6, 13. Schenk, F. (1910). Beitr. z. klin. Chir. 67, 316. Schenk, F. (1927). Z. Geburtsh. Gynak. 91, 483. Severinghaus, A. E., Engle, E. T. and Smith, P. E. (1932). Sex and Internal Secretions, p Baltimore: Williams and Wilkins. Soli, U. (1909). Arch. ital. Biol. 52, 353. Tandler, J. and Gross, S. (1913). Die biologischen Grundlagen der sekundaren Gewchlecht8- charactere. Berlin: J. Springer. Tescione (1904). Arch. ital. Ginec. Zchwetadse, J. (1927). Bus8k. Klin. 7, 621.

31 THE JOURNAL OF PHYSIOLOGY, VOL. 79, No. 1 PLATE I Fig. 1. Adrenal of a normal male rat, aged 87 days. H. and E. x 50. Fig. 2. Adrenal of a male rat which was castrated at 21 days and killed at 85 days. H. and E. x 50. Fig. 3. Adrenal of a male rat which was -castrated at 80 days and killed after 1 week. H. and E. x 50.

32 PLATE II THE JOURNAL OF PHYSIOLOGY, VOL. 79, No. 1 :-':. Fig. 4. Adrenal of a female rat which was Fig. 5. Adrenal of a female rat which was spayed at 80 days and killed after'l week. spayed at 80 days and killed after H. and E. x 0I. 6 weeks. H. and E. x 50. Central portion of the thyroids of a normal rat aged 136 days. H. and E. x 217. Fig. 6. Fig. 7. Central portion of the thyroid of a male rat castrated at 80 days and killed at 136 days. H. and E. x 217.