612. 45 : 612 . 616. 3

CHANGES I N THE ADRENALS OF GONAD- ECTOMISED MALE AND FEMALE RATS PRODUCED BY PROLONGED INJECTIONS OF SEX HORMONES

KATHLEEN HALL Prom the Lister Institute, London

(PLATES X AND XI)

THE present paper describes the histological changes in the adrenals of gonadectomised male and female rats produced by injections of different sex hormones for a prolonged period of about 100 days. The changes which occur in the weights of the organs, including the adrenals, in the majority of these rats have been recorded in previous papers (Korenchevsky et al., 1939 a and b) and will not be described here. An additional 30 female rats from unpublished experiments are included. The changes obtained after injections of male hormones alone are compared with those previously described in males (Hall and Korenchevsky, 1937-38) in experiments of much shorter duration (mostly 3 weeks).

HISTORICAL

Effects of &ration on the adrenals

A review of the literature and the results of our own experiments on males were given in the earlier paper (Hall and Korenchevsky). More recently, the hypertrophying effect of castration on the adrenals of rats has been confirmed by Chiodi (1938), while Howard (1937-38) found no structural changes in the cortex after castration.

Effects of ovariectomy on the ccdrenals

Histologically Andersen and Kennedy (1933), in the rat, found dcgenerative changes in the reticular zone and decreased, lipoid content. Zalesky (1934), in the ground squirrel, described intense destruction in the reticular zone and its replacement by a fibrous ring around the medulla. An excellent summary of the literature on changes in size or weight of the adrenals after ovarjeotomy has been given by Blumenfeld (1939), to which paper readers are referred. Briefly, he records that most authors describe cnlttrgement of thc gland in the rabbit, decrease in size in the guinoa-pig and rat and no change in the mouse. Blumenfeld himself found in the rat a decrease in width of tho cortex, especially of the zona fasciculata (duo chiefly to decrease in size of the cells), increased cell destruction and increased

75

76 K . HALL

pigmentation in the zona reticularis and a general reduction in lipoid content of the cortex. Silvestroni (1939) has described a slight increase in the number of lipoid droplets in the zona fasciculata.

Effect of male hormones

Hall and Korenchevsky found that different male hormones when injected in various doses for 3 weeks into castrated rats produced a compara- tively lasting recovery to or towards normal, both in weight and in histological structure of the adrenals. Pathological features, however, were also observed -decrease to below normal in the width of the zones and size of the cells and decreased amount of lipoid. Silvestroni found a decrease in lipoid content in females after testosterone injections.

Disappearance of the X zone in both sexes after injections of testosterone propionate has been recorded by Cramer and Horning (19374 and Starkey and Schmidt (1938) and in castrated males after administration of male hormones by Deanesly and Parkes (1936-37).

Rat.

Mouse.

Effect of mstrogem

Rat. Histological changes described in the rat after estrogen ad- ministration include widening of the cortex, especially the zona fasciculata, and hypertrophy of its cells (Ellison and Burch, 1936 ; Silvestroni, 1939 ; Bacsich and Folley, 1938-39); sometimes cystic adenoma in the cortex (McEuen, Selye and Collip, 1936) ; increased mitoses (Ellison and Burch, 1936) ; changes in vacuolation and lipoid content-an increase by del Castillo and Sammartino (1937) and Silvestroni (1939), a decrease by Selye, Collip and Thomson (1934-35).

In the male, Burrows (1936) described the reappearance of the X zone and extensive degenerative changes in the cortox after prolonged administration of estrogens. Cramer and Horning ( 1 9 3 7 4 recorded that the X zone does not disappear in the female and (1936, 193723) described degenerative changes in the cortex in both sexes. Lacassagne and Raynaud (1937) found that involution of the X zone is not modified by estrogens. Danner (1938-4-6 months’ treatment) described great degeneration of the zona fasciculata and zona reticularis but not of the zona glomerulosa in both sexes.

Mazer, Israel and Alpers (1936) record hypertrophy of the outer cortical cells and medullary invasion by groups of mid-zonal cortical cells.

Mouse.

Rabbit.

TECHNIQUE

With the exception of 30 females, all the animals used were from the long duration experiments previously described (Korenchevsky et al., 1939 a and b) and details of the technique can be obtained from these papers. The adrenals of 88 male and 101 female rats were investigated. Thirty male and 21 female intact or gonadectomised animals were kept as controls; the others were gonadectomised and injected with the various hormones for an average period of 87 days for females and 105 days for males. Gonadectomy was performed at the age of 21-25 days. All normal female control rats were killed when in dicestrus. The male hormones were injected five times a week, progesterone six times a week and cestradiol esters three times a week (except the dose of 0.2 mg. given once a week).

The methods employed in the microscopic examination of the adrenal glands have already been described (Hall and Korenchevsky). Paraffin

The general arrangement of the experiment is shown in the table.

ADRENAL CHANCES DUE T O SEX HORMONES 77

NO. of rats In group

TABLE

Sumrnary of experiments

4zgfEf adrenals

Hormone and wcckly dose injected

9

12

15

8 21 22

6

Normal controls injected with

Gonadectomised controls injected

CEstradiol D* or B.B.,* 0-018-0.09

CEstradiol D, 0.2 mg. . Androgens,? 2.25-7.5 mg. . . Androgens,? 2.25-7.5 + cestradiol

Testostorone P,* 2.25 + cestradiol

sesame oil

with sesame oil

mg.

D or B.B., 0.018-0.09 mg.

71

70

79

59 59 85

64 D, 0.2 mg.

Testosterone P, 2.25 + aostradiol I), 0-2 + progesterone, 4.5 mg.

Males

No. of rats in group

16

14

9

... 29 20

...

...

Average weight of adrenals

55

75

64

... 59 74

...

...

Females

I 62

* D = dipropionate ; B.B. = 3-benzoate-17-n-butyrate ; P = propionate. t Thc androgens used were as follows (the figures in brackets representing the weekly dosc in mg.

injected) :-Androsterone (7.6), dehydroandrosterone (7.6), testosterone (2.25-7-51, testosterone propionate (2.26-7.5), testosterone dipropionate (7.6).

sections were stained with Ehrlich’s haematoxylin and eosin and when necessary with Masson’s trichromic stain for fibrous tissue. Frozen sections were stained for lipoids with Scharlach R.

RESULTS Efleect of castration

The changes produced by castration were similar to those already described (Hall and Korenchevsky), i.e. hypertrophy of the gland chiefly due to increase of the zona fasciculata and zona reticularis, enlargement of the cells in these zones, hyperBmia in the zona reticularis, slight narrowing of the. zona glomerulosa, and a slight increase in the number of lipoid droplets in the cortex. The demarcation zone, however, which we previously described as disappearing after castration, was in these experiments absent in comparatively few “ castration ” adrenals, but in most of them it was narrower and less conspicuous than in the normal controls.

Eflect of ovariectomy (jigs. 2 and 6)

In keeping with the absence of any constant change in size of the glands, there was no hypertrophy of the zona fasciculata or zona reticularis. As in males, there was a slight decrease in the width of the zona glomerulosa and in the size of its cells. The demarcation zone might! be present or absent in both spayed and normal controls and when present was usually narrow. No constant

78 K . HALL

changes were observed in the zona fasciculata, but in the zona reticularis of most of the spayed animals there was increased vascularisation and hyperaemia and an increased number of atrophic cords consisting of atrophic cells (compare figs. 2 and 1). In the deeper layers of the reticularis bordering on the medulla, there appeared, exceptionally in normal animals and occasionally in spayed animals, small groups or islets of cells which were slightly larger than the majority of reticularis cells, which were not separated into columns by blood vessels and which contained fewer or no lipoid droplets. There was no change in the lipoid droplet content of the zona glomerulosa and zona fasciculata, but, in a few cases only, depletion was noticed in the zona reticularis.

Effect of mtradiol dipropionate or 3-benzoate-17-n-butyrate

The effect of estradiol esters on the adrenals was similar in both cases. Except with the largest dose of 0.2 mg. their effect on the size of the adrenals was indefinite and inconstant (see table). All the doses, however, produced changes in the histological structure, the parts most affected being the zona reticularis and zona glomerulosa. Except for increased vascularisation, no changes were noticed in the medulla.

Hyperzemia and degeneration of the cells of the reticular zone constituted the most pronounced feature and often resulted in their almost complete atrophy (figs. 3 and 7). The nuclei of these cells were degenerated and often shrunken and pyknotic. The remnants of these cells sometimes coalesced to form what appeared at first sight to be single much elongated and very narrow cells, containing in some cases up t o ten closely packed nuclei. In extreme cases this process resulted in the disappearance of the columns of reticularis cells and their replacement by either fibrous trabeculae separated by wide blood vessels or even in the inner part of the zone by a compact ring of fibrous tissue surrounding the medulla (fig. 8). Danner (1938) has described a somewhat similar process of degeneration in the cortex of the mouse adrenal after estrogen treatment. The changes extended, however, into the zona fasciculata, whereas in our experiments they were confined to the zona reticularis.

In many adrenals of our rats there were groups or islets of large rounded cells, usually with well marked nuclei and eosinophilic cytoplasm (figs. 3, 4 and 7). These cells were usually larger than the ordinary reticularis cells and were not separated into columns by blood vessels. In the majority lipoid droplets were absent or few and very small and the cytoplasm stained diffusely with Scharlach R. In some cases these islets were sufficiently large and numerous to form an interrupted zone between reticularis and

PLATE X

FIU. 1.-Normal uninjected female rat. From above downwards : zona glomerulosa, separated by narrow demarcation zone from the zona fasciculata, zona reticitlarib, part of medulla.

FIU. 2. -Spayed uninjected control rat. Glornerulosa less clearly demarcated from Rat 3 10 g.,

Rat 300 g., adrenals 77 mg. x 53.

fasciculata. Some hyperseniia and cellular atrophy in reticularis. adrenals 62 mg. X 53.

FIU. 3 .4payed rat injected with cestradiol dipropionate (0 .2 mg. per week). Narrow glomerulosa ; extensive hyperzemia and cellular atrophy in reticularis ; bottom left, islet of " reticularis boundary " cells. Rat 170 g., adrenals 57 mg. Period of injections 91 days.

FIG. 4.Spayed rat injected with cestradiol dipropionate (0.018 mg. per week). Cross section of adrenal at low magnification showing changes similar to those in fig, 3, including several islets of " reticularis boundary " cells (dark patches around medulla). Rat 215 g., adrenals 76 mg. Period of injections 86 days. x 17.

FIU. 5.-Spayed rat injected with mstradiol dipropionate (0.09 mg. per week) +testosterone dipropionate (7 .5 mg. per week). Neutralising effect of male hormone on histological changes produced by cestradiol. The gland is almost normal. Rat 250 g., edrenals 96 mg. Period of injections 63 days. x 53.

x 53.

JOURNAL Oh’ 1’BTHOLOGY-VOr~. LI

ADRENAL CHANGES DUE TO SEX HORMONES

BIG. 1.

FIG. 3.

FIG. 2.

FIG. 4.

PLATE X

FIG. 5.

A D R E N A L CHANGES DUE T O S E X HORMONES 79

medulla (fig. 4). Exceptionally, a few much smaller islets of these cells were found in our normal or gonadectomised animals but were never conspicuous. They appear to be similar to those recorded in female rats by Andersen and Kennedy and probably also to those observed in normal and castrated rats by Howard. This author even suggests some relation of these cells to the X zone cells in mice. Bacsich and Folley have described these cells -which they designated Y cells-as being especially numerous in lactating rats and less numerous in such rats injected with estrogens. Dr Korenchevsky has suggested that the name " reticularis boundary ') cells would be appropriate, as they are situated on the boundary between cortex and medulla and derived from the reticularis.

In several adrenals the zona glomerulosa was also affected. In extreme cases it became exceedingly narrow and its cells were reduced in size and irregularly disposed. The closely packed nuclei made the zone specifically conspicuous. In some cases hyperemia appeared in this zone and the number of cells was decreased. In many adrenals, especially with the two lower doses, lipoid droplets had completely disappeared from the zona glomerulosa and in most rats there was also a reduction in their number in the fasciculata and reticularis, especially with the lower doses.

(Estrogenic hormones apparently have the effect of extending and increasing the comparatively mild changes which have already appeared in the reticularis and glomerulosa of the glands as a result of gonadectomy. The pathological changes were produced in similar degree by all the doses used, with the exception of the more extreme fibrous changes in the reticularis, which appeared chiefly with the largest dose.

Eflect of male hormones

In general all the male hormones caused a return to or towards normal of the pathological changes produced by gonadectomy , but when the result of the present experiments are compared with those of our previous experiments of much shorter duration (Hall and Korenchevsky) it appears that prolongation of the period of injections did not, as might have been expected, cause greater changes. In fact the effect of the hormones was in some respects less pronounced, as though some neutralising mechanism had been operating.

In males, all the male hormones except testosterone dipropionate caused a return to or towards normal in the size of the adrenals and in the width of the cortical zones. The abnormal decrease in size which in our previous experiments was produced by androstcrone was not observed. The effect of the male hormones on females was

80 K . HALL

to produce adrenals of similar size to those of normal uninjected inales or of castrated males injected with the same hormones. The normal adrenal gland is generally larger in females than in males, but treatment of females with male hormones causes the dis- appearance of this sex difference in size.

In both sexes the zona glomerulosa became wide and much more conspicuous, a well marked demarcation zone usually appeared and the degenerative changes and hyperaemia were less pronounced or had disappeared from the zona reticularis. With large doses of testosterone propionate and dipropionate, however, a certain amount of hyperzemia appeared in the zona fasciculata. In our previous short-duration experiments this condition appeared after injections of androsterone (see fig. 6, pl. I ; Hall and Korenchevsky), but not of testosterone propionate.

All the male hormones used tended to reduce the number of lipoid droplets in the cortex, but, except with the large doses of testosterone propionate and dipropionate-which caused consider- able depletion similar to that shown in fig. 11, pl. I1 (Hall and Korenchevsky )-the effect on the lipoid content was less pronounced than in our earlier short-duration experiments. Androsterone, dehydroandrosterone, testosterone and the smaller dose of testo- sterone propionate caused only a slight decrease of lipoid in the zona fasciculata and zona reticularis, while there was in some cases even a slight increase in the zona glomerulosa (compare with fig. 10, pl. I1 of the earlier paper).

Effects of combined injections of mstradiol dipropionate and male hormones

Histologically most of the male hormones were able to prevent at least some of the pathological changes produced by estrogens. Dehydroandrosterone was found to be the weakest of the male hormones in this respect. Androsterone and the small dose of testosterone had a stronger but still weak neutralising action on the cestradiol changes. The adrenals of rats injected with these hor- mones showed hyperzmia of the zona reticularis and in some cases degeneration of cells similar to, though often of a lesser degree than, that produced by cestradiol alone ; sometimes they showed islets of " reticularis boundary " cells and a narrow zona glomeruiosa with small cells.

The small doses of testosterone propionate and the large dose of testosterone partly neutralised while the large dose of testosterone propionate and in some cases of dipropionate (fig. 5) almost com- pletely prevented the histological changes produced by cestradiol, so that in all but size and weight the adrenals of these animals were similar to those of rats injected with the male hormones alone. With the combined injections with cestradiol esters all

PLATE XI

FIG. 6.-Spayeduninjected control rat. Lower part of zona reticularis in cross section at high magnification, with a few cells (light) of medulla at bottom. The blood veasels separating the cords of reticularis cells are slightly dilated as compared with those of non-spayed control rats. Rat 340 g., adrenals 62 mg. x 540.

FIG. 7.-Spayed rat injected with cestradiol dipropionate (0.018 mg. per week). Lower part of zona reticularis in cross section, showing medium effect of estrogen. Hyperamia is much more pronounced and cellular atrophy i s present in most of the cell cords, some of' which are reduced to narrow fibrous trabeculie. In several places the cells-often atrophic-have coalesced to form multinucleated syncytia. Adjoining the medulla (light cells a t the bottom) is part of an islet of " reticularis boundary " cells (dark). Rat 215 g., adrenals 76 mg. Period of injections 86 days. x 540.

Fra. 8.-Spayed rat injected with cpstradiol dipropionate (2 mg. per week). Low-er part of zona reticularis in cross section, showing strong effect of estrogen. The cords of reticularis cells have been replaced by fibrous tissue which in the lower part forms a fibrous ring surrounding the medulla (a few cells of which are shown a t the bottom). Period of injections 146 days. x 540.

Rat 178 g., adrenals 5 1 mg.

JOURNAL OP PATHOLOGY-VOL. LI

ADRENAL CHANGES DUE TO SEX HORMONES

FIG. 6.

FTQ. 8.

PLATE XI

FIG. 7.

ADRENAL CHANGES DUE T O SEX HORMONES 81

the male hormones except dehydroandrosterone caused a consider- able decrease in the lipoid droplet content of the cortex.

The addition of progesterone to the injections of testosterone propionate and cestradiol dipropionate into spayed rats (see table) did not alter the effect on the weight and size of the glands. Histo- logically in some rats the effect of the cestradiol predominated (hyperaemia and degeneration of the zona reticularis, decrease in width of the zona glomerulosa and in the size of its cells). In others the cortical zones appeared normal. The lipoid content of the zona fasciculata and zona reticularis was decreased in some glands only; that of the zona glomerulosa was normal or even increased.

SUMMARY 1. The effect on the adrenals of gonadectomy and of prolonged

injections (about 100 days) of different male and female sex hormones was studied in 88 male and 101 female rats.

2 . Gonadectomy in males caused hypertrophy of the zona fasciculata and zona reticularis of the adrenals, hyperaemia of the zona reticularis, slight narrowing of the zona glomerulosa and slight increase of lipoid droplets.

3. Gonadectomy in females produced no constant change in the width of the zona fasciculata or zona reticularis, but the zona gloinerulosa was slightly narrower, and some hypermnia and cellular atrophy were present in the zona reticularis.

4. In both sexes estrogens produced no constant change in size, but the pathological changes included cellular atrophy, fibrous degeneration and hyperzemia in the zona reticularis, the appearance of islets of " reticularis boundary '' cells, considerable decrease of the zona glomerulosa and of its cells, and in most cases a decrease in lipoid content of all cortical zones.

5. In both sexes all the male hormones caused a return to or towards normal of the size and histological structure of the adrenals, but the testosterone esters caused lipoid depletion and slight hypersniia in the zona fasciculata.

6. As compared with experiments of shorter duration, prolonga- tion of the injections of male sex hormones did not produce greater but rather less pronounced changes, as though some neutralising mechanism had developed.

7. In both sexes, when male hormones were injected simultane- ously with estrogens, the male hormones prevented, partially or even completely, the pathological changes produced by acstradiol, dehydroandrosterone having the weakest and testosterone esters the strongest powers in this respect.

I should like to thank Dr V. Korenchevsky for much helpful advice and A grant from the Modical Research Council has enabled me to criticism.

JOURI . OF PATH.-VOL. LI F

82 K . HALL

carry out the work. My thanks are also due to Messrs Organon Ltd. for kindly supplying progesterone and to Messrs Ciba Ltd., in particular to Dr K. Miescher, for the other hormones.

REFERENCES

ANDERSEN, D. H., AND KEN-

BACSICH, P., AND FOLLEY, S. J. BLUMENFELD, C.M. . . . BURROWS, H. . . . . . DEL CASTILLO, E. B., AND

CHIODI, H. . . . . . . CRAMER, W., AND HORNING,

NEDY, H. S.

SAMMARTINO, R.

E. s. ,> , 1 9 1 ,

1, 3, ,+ 7 ,

DANNER, M. . . . , . . DEANESLY, R., AND PARKES,

ELLISON, E. T., AND BURCH,

HALL, K., AND KORENCHEVSKY,

HOWARD, E. . . . . . . KORENCHEVSKY, V., HALL, K.,

KORENCHEVSKY, V., HALL, K.,

LACASSACNE, A., AND RAY-

MAZER, C., ISRAEL, S. L., AND

MCEUEN, C. S., SELYE, H., AND

SELYE, H., COLLIP, J. B., AND

SILVESTRONI, E. . . . . STARKEY, W. F., AND SCHMIDT,

ZALESKY, M. . . . . .

A. S.

J. C.

V.

AND BURBANK, R.

AND ROSS, M. A.

NAUD, A.

ALPERS, B. J.

COLLIP, J. B.

THOMSON, D. L.

E. C. H., Jr.

1933. J. Physiol., Ixxix, 1.

1938-39. J . Anat., lxxiii, 432. 1939. Endocrinology, xxiv, 723. 1936. This Journal, xliii, 121. 1937.

1938. Ibid., xiv, 246. 1936. Lancet, i, 247.

Rev. Sac. argent. Biol., xiii, 455.

1937a. Ibid., i, 1330. 19376. This Journal, xliv, 633. 1938. KZin. Wschr., svii, 658. 1936-37. Quart. J. Ezp. Physiol., xxvi,

1936. Endocrinology, xx, 746.

1937-38. J . Physiol., xci, 365.

393.

1937-38. Amer. J. Anat . , Ixii, 351. 1939a. Biochem. J., xxxiii, 372.

1939b. Ibid., xxxiii, 213.

1937. C.R. SOC. biol., cxxiv, 1186.

1936. Endocrinology, xx, 753.

1936. Lancet, i, 775.

1934-35. Proc. SOC. Exp. Biol. and Med.,

1939. 1938. Endocrinology, xxiii, 339.

xxxii, 1377. Arch. Ist. biochim. ital., xi, 71.

1934. Anat . Rec., lx, 291.