182

EXPERIMENTAL-HISTOLOGICAL STUDIES ON THE CHANGES IN THE PITUITARY OF THE MOUSE FOLLOWING ADMINISTRATION OF ANTERIOR LOBE EXTRACT (PRAEHORMON)

TATsuo Aor

Department of Anatomy, Nagoya University School of Medicine ( Director : Prof. Kazumaro Yam ada)

In spite of extensive developments in hormone-therapy in recent years, it seems very difficult to determine the correct therapeutical dose of anterior pitui­ tary hormone for all indications. It can be assumed, moreover, that both ex­ cessive and insufficient administrations have been made frequently in the appli­ cation of this hormone. Consequently, it is conceivable that the hormone-balance of the body receiving such hormone treatment will be more or less affected by such undesirable administrations, and that such hormone-unbalance may result in significant functional alterations in certain endocrine glands. Therefore, it will be of high significance to investigate the effectiveness of this hormone which has an action on the hypophysis itself. For the purpose of clarifying the functional state of the hypophysis in cases receiving this hormone a study of histological changes in the pituitary of mouse injected with an anterior lobe extract was undertaken. At present, there are several reports concerning the problem mentioned above, in mice,8 > rats4 >5 > and guinea-pigs."> These reports, however, do not contain details sufficient for an understanding of the secretory activity of the pituitary. The purpose of the present paper is to serve as an introduction to further information on this problem.

MATERIALS AND METHODS Seventy-seven mature male mice were subjected daily to subcutaneous in­ jections of an anterior lobe extract (Praehormon) for one to twenty-eight days. Half of them were injected with 0.1 rat unit of Praehormon per day and the other with 1.0 rat unit. All animals were killed on the day following the last treatment and their pituitaries were removed. In the present study, Zenker's, Reily's, Kolster's, Regaud's and Ciaccio's fluids were employed as fixatives. After fixation the pituitaries were dehydrated in alcohol and embedded in paraffin by routine procedures. Materials were sectioned at 4 or 6 micra serially and were stained chiefly with Hansen's hematoxylin and and with Heidenhain's iron hematoxylin. In some sections, Gomori's rapid one step trichrome stain 7> and Heidenhain's azan stain were also employed.

Received for publication July 4, 1956. MOUSE HYPOPHYSIS FOLLOWING PRAEHORMON ADMINISTRATION 183

RESULTS AND DISCUSSION

A. Proper capsule The proper capsule of the pituitary showed an almost similar appearance as those of normal mice.

B. Anterior lobe The arrangement of the parenchymal and border cells and the interstitial connective tissues did not appear to show any alterations, when compared with the normal aspect.

1) Blood capillary In the peripheral blood vessels, sinusoidal enlargement and hyperaemia were frequently observed and indicated gradual increase with increase in number of administrations (Figs. 1 and 4). These observations agree with those of Haterius and Charipper,s) while Baniecki 4 ) and Collip, Selye and Thomson 'l reported that intense hyperaemia or bleeding is observed in the of the rat treated with an anterior lobe extract. These different results are due probably to the dose of anterior lobe extract used. Further, it is probably justifiable to consider that these changes in the blood capillaries occur as an autoregulation phenomenon resulting from a functional alteration of the anterior pituitary due to the treatment. Consequently I can not support the results of Franck 6) who did not recognize an hyperaemia with various doses of the extract.

2) Chromophobe Chromophobe cells increased in number with progress of the experiment, in general. Typical chromophobe cells (Fig. 1) showed a tendency to reduction in number, while the number of chromophobe cells containing dark or irregular­ shaped nuclei (Fig. 1) increased in proportion to the succession of experiment. The possessing a slightly dark nucleus and scanty light cytoplasm (Fig. 3) showed, even though not so marked, an increase in number with progress of the experiment. This chromophobe cell has mainly a round or oval nucleus which is closely similar to that of an eosinophile cell, and can be accepted to be a mutually transitional form between the chromophobe and eosinophile cells. In addition, chromophobe cell containing a light vesicular nucleus and scanty light cytoplasm and showing a distinct cell boundary (Fig. 2) was observed somewhat abundantly than in the normal. The chromophobe cell containing a nucleus in which nodular or droplet-like chromatin granules are irregularly distributed (Fig. 3 and 7) showed some in­ crease with continuation of treatment. These nuclei were generally ovoid, elon­ gated or rarely irregular-shaped, and sometimes two of them were located side by side. These nuclei will be designated as "droplet-nucleus" for descriptive purposes. In regard to chromophobe cells, Haterius and Charipper 5l described that definite variation was not observed, but the present results did not agree with 184 T. AOI

their data. The transformation of nuclei described by Baniecki 4l was also ob­ served in the present experiment. Collip, Selye and Thomson 5l reported the appearance of extremely large cells containing eccentrically situated nuclei or large vacuoles in the cytoplasm, and Baniecki pointed out vacuolization of nuclei and an increase in amount of chromatin. In the present observations, however, these changes could not be recognized. On the other hand, an increase in num­ ber of chromophobe cells was observed in the present experiment as in Franck's data, especially in animals injected with 0.1 R.U. per day of Praehormon.

3) Basophile cell It is possible to classify basophile cells into three types, viz. typical basophile, B-1 and B-2 cells. The typical basophile cell: The cell contains a large nucleus showing a delicate chromatin network and abundant cytoplasm or am­ phophilic (Figs. 1 and 3). The cell showed gradual decrease in number with progress of the experiment, and especially in the middle stage the decrease was marked. On the contrary there appeared an increase in number of the cell towards the end. The B-1 cell: The cell contains a relatively large, dark nucleus with a delicate chromatin network and with comparatively distinct karyosomes, and somewhat abundant cytoplasm indicating feeble stainability (Fig. 3 and 5). The number of this cell at the early stage of experiment showed an increase in number. Later, with progress in administration the number of this cell de­ creased gradually accompanied by a temporary increase. The B·2 cell: This cell contains a large, light nucleus denoting somewhat coarse chromatin network and little cytoplasm which is difficult to stain with various dyes (Fig. 3). A slight increase in this cell was found only in the middle stage. With a decrease in number of typical basophile cell, there was an increase in number of B-1 cell and a slight increase of B-2 cell. The marked decrease in typical basophile cell was followed by an increase of B-2 cell, and later by B-1 cell. Toward the end of this experiment, increase of typical basophile cell and decrease of B-1 and B-2 cells were observed. From these findings, it can be suggested that the typical basophile and chromophobe cells can pass mutually through the B-1 and B-2 cells. The decrease in number of typical basophile cell observed in the present in­ vestigation agrees with the results of Franck who found a decrease in number of basophile cell in his shorter term experiment. However Franck, so also Haterius and Charipper and Baniecki, found no changes in basophile cells in his longer term experiment. In the present data, there were also some differences in the tendency to decrease in number of basophile cell according to the dose of Praehormon, and this will depend partly on a decline in biological response of an individual or partly on the occurrence of an autoregulatory function of the anterior lobe. From this, it may be proper to consider that, under certain circumstances, the typical basophile cell can show almost the same appearance as that in normal animals. MOUSE HYPOPHYSIS FOLLOWING PRAEHORMON ADMINISTRATION 185

Collip, Selye and Thomson observed basophile cell with a nucleus situated eccentrically in the cytoplasm and extremely large cell containing large vacuoles in the cytoplasm. These cells, however, were not found in the present obser­ vations. Franck found an increase in number of small basophile cell and of basophile cell containing few granules in the cytoplasm in his shorter term ex­ periment. It is probable that these cells will correspond respectively to my B-2 and B-1 cell, and the appearance of these cells agrees with that in the present experiment.

4) EosinoPhile cell The typical eosinophile cell containing a round or oval, light vesicular nucleus and somewhat coarse, acidophilic granules in the cytoplasm (Fig. 2) showed gradual decrease in number with increase in number of administration. On the contrary, there could be observed a gradual increase in number of cells containing irregularly shaped (Fig. 4), dark (Fig. 2), metachromatic (Figs. 4 and 7) or pyknotic nucleus (Figs. 4 and 7). Mitotic figures of eosinophile cells were more abundant than in normal (Fig. 3). Eosinophile cell containing the "droplet-nucleus" mentioned above and cyto­ plasm which stain faintly with eosin showed an increase in number (Figs. 3 and 6). The cell appeared more frequently when mitoses of eosinophile cells were abundant. Sometimes two of the cells appeared side by side as was described for the chromophobe cell. Although there has not been any report on the "droplet-nucleus" in the anterior lobe, it is probable that the cell containing such nucleus will have an intimate relationship with a certain special secretory activity or with a special mitotic phase. Changes in the cytoplasmic granulation of eosinophile cells were found in the present experiment. Cells indicating rather indistinct, minute granulation in the cytoplasm showed a prominent increase in number (Fig. 5). Eosinophile cells containing moderate-sized or large cytoplasm (Fig. 7), those presenting a negative image of Golgi's apparatus in the cytoplasm (Fig. 5) and those indicating an indistinct cytoplasmic granulation which stain homo­ geneously reddish with eosin (Fig. 4) decreased in number. On the other hand, eosinophile cells containing little cytoplasm (Fig. 7), those possessing feebly cytoplasm (Fig. 1), those indicating irregular­ ly acidophilic, minute granulation in the cytoplasm (Fig. 4) and those containing a small quantity of slightly acidophilic cytoplasm (Figs. 2 and 4) showed an in­ crease in number. The last, moreover, can be accepted to be a transitional form between the chromophobe and eosinophile cells. As mentioned above, although eosinophile cells showed various alterations in form in the present experiment, the marked appearance of large cells reported by Haterius and Charipper was not proved. I also failed to find extremely large cells containing large vacuoles in the cytoplasm such as was pointed out by Collip, Selye and Thomson. The present results agree with those of Baniecki who found an increase in number of eosinophile cells containing dark or irregular­ shaped nucleus. He also recognized an increase in eosinophile cells, while a tendency to decrease in number of the cells was noted in the present results. 186 T. AOI

Franck has reported an increase in number of small eosinophile cells, faintly acidophilic cells and eosinophile cells having little cytoplasmic granules, in ac­ cordance with decrease in number of typical eosinophile cell. It is conceivable that his small eosinophile cell is identical with the cell showing slightly acido­ philic cytoplasm, or with the cell indicating irregularly , minute granulation in the cytoplasm, of my data. Thus the present results agree with those of Franck regarding the appearance of eosinophile cells.

5) Border cell Regarding the border cells, no definite distinctions could be found from those of the normal.

6) Summary In the present experiment, the anterior lobe tissues showed certain retro­ gressive changes in general. These changes appeared more promptly and severely in the group of 1.0 R.U. administration than in the 0.1 R.U. group, while they did not become intense in proportion to the number of injection. On the con­ trary, in the 0.1 R.U. group these changes which occured slightly in the early stage of the experiment became severe gradually. Thus in cases in which the total amount of Praehormon injected was equal, there appeared more prominent changes in the 0.1 R.U. group than in the 1.0 R.U. group. Namely, the adminis­ tration dosage of Praehormon did not always run parallel with the degree of histological changes in the anterior lobe. These facts may suggest that the histological changes described above will be influenced by the autoregulatory function of the anterior pituitary, the production of antihormone and the decline in receptivity of an individual to Praehormon.

C. There have been only a few reports concerning the pars intermedia in ex­ periments of the anterior lobe extract administration. Baniecki and Franck reported no findings in their experiments. In the present examination, no dis· tinct change in the pars intermedia appeared. In parenchymal cells, however, cells containing metachromatic or pyknotic nucleus and light, scanty cytoplasm and those containing so-called "nucleus-vacuole" and little cytoplasm showed slight increase in number.

D. Pars nervosa As regards the pars nervosa, Baniecki did not observe any definite change, while Franck reported an increase in number of basophile colloid. In the present investigations, parenchymal cells, glia-like cells, glia-like fibers, connective tissues and colloidal substances showed almost the same ap· pearance as in normal mice. But the blood capillary showed gradually a slight increase in sinusoidal enlargement and hyperaemia, proportional to progress of experiment (Fig. 8). The hyperaemia in the was found mostly to correspond with that in the anterior lobe. MOUSE HYPOPHYSIS FOLLOWING PRAEHORMON ADMINISTRATION 187

CONCLUSION The anterior pituitary of the mouse injected with an anterior lobe extract ( Praehormon) showed in general retrogressive changes. These changes appeared more promptly and severely in the larger dose administration group than in the smaller one, while they did not become intense in proportion to the number of injection. On the contrary, in the smaller dose group these changes occurred slightly in the early stage of the experiment and became more marked gradually. Thus in cases in which the total amount of Praehormon injected was equal. there appeared more prominent changes in the smaller dose and longer term administration than in the larger dose and shorter term group. Namely, the degree in histological changes in the mouse anterior pituitary did not always run parallel with the administration dosage of Praehormon.

ACKNOWLEDGEMENT I wish to express my gratitude to Prof. Dr. K. Yamada for his guidance and criticism throughout the course of this investigation.

REFERENCES

1. AO!, T. ]. Nagoya med. Ass. 68: 778, 1954. 2. AO!, T. ]. Nagoya med. Ass. 69: 353, 1955. 3. AO!, T. ]. Nagoya med. Ass. 69: 479, 1955. 4. BANIECKI, H. Arch. Gyniikol. 149: 478, 1932. 5. CoLLIP, ]. B., H. SEL YE AND D. L. THOMSON. Proc. Soc. Exp. Bioi. and Med. 31 : 682, 1934. 6. FRANCK, S. Acta path. et microbial. scand. 14: 339, 1937. 7. GOMORI, G. Am. ]. Clin. Pathol. 20 : 661, 1950. 8. HATERIUS, H. 0. AND H. A. CHARIPPER. Anat. Rec. 51: 85, 1931. 188 T. AOI

EXPLANATION OF FIGURES FIG. 1. Anterior pituitary of the mouse injected with 0.1 R.U. Praehormon for 21 days. Fixed in Helly's fluid, and stained with Heidenhain's iron-hematoxylin. x800. Sinusoidal enlargement and hyperaemia in peripheral blood vessels (a), typical chromophobe cell (b), chromophobe cells containing irregular-shaped nucleus (c) and slightly dark nucleus (d), typical basophile cell (e), B-2 cell (j) and eosinophile cell possessing feebly acidophilic cytoplasm (g). FIG. 2. Anterior pituitary of mouse injected with 0.1 R.U. Praehormon for 4 days. Fixed in Helly's fluid, and stained with Heidenhain's iron-hematoxylin. x 800. Chromophobe cell containing a light vesicular nucleus and scanty light cyto­ plasm (a), typical eosinophile cell (b), eosinophile cells containing light vesicular nucleus (c), slightly dark nucleus (d) and dark nucleus (e), eosinophile cell con­ taining a light vesicular nucleus and a small quantity of slightly acidophilic cytoplasm (/). FIG. 3. Anterior pituitary of mouse injected with 1.0 R.U. Praehormon for 14 days. Fixed in Helly's fluid, and stained by hematoxylin-eosin stain. x 800. Chromophobe cell possessing slightly dark nucleus and scanty light cytoplasm (a), chromophobe cell containing nucleus in which nodular or droplet-like chro­ matin granules are distributed irregularly (b), typical basophile cell (c), B-1 cell (d), B-2 cell (e), mitotic figure of eosinophile cell (/), eosinophile cell contain­ ing "droplet-nucleus" (g). FIG. 4. Anterior pituitary of mouse injected with 0.1 R.U. Praehormon for 7 days. Fixed in Ciaccio's fluid, and stained with Heidenhain's iron-hematoxylin.. x 800. Sinusoidal enlargement and hyperaemia in peripheral blood vessels (a), eosino­ phile cells containing irregularly shaped nucleus ( b and c), eosinophile cell indi­ cating irregularly acidophilic, minute granulation in the cytoplasm (d), eosinophile cells possessing metachromatic or pyknotic nucleus (e), eosinophile cell indicating an indistinct cytoplasmic granulation which stain homogeneously red with eosin (j), eosinophile cell containing slightly dark nucleus and small amount of slightly acidophilic cytoplasm (g). FIG. 5. Anterior pituitary of mouse injected with 1.0 R.U. Praehormon for 21 days. Fixed in Helly's fluid and stained by hematoxylin-eosin stain. x 2 000. B-2 cells (a), eosinophile cells containing a round or oval nucleus (b), typical eosinophile cell with a negative image of Golgi's apparatus in the cytoplasm (c), eosinophile cell indicating rather indistinct (d) and very indistinct (e) granu­ lation in the cytoplasm. FIG. 6. Anterior pituitary of mouse injected with 1.0 R.U. Praehormon for 28 days. Fixed in Helly's fluid, and stained by hematoxylin-eosin stain. x 2 000. Several eosinophile cells containing "droplet-nucleus." FIG. 7. Anterior pituitary of mouse injected with 0.1 R.U. Praehormon for 21 days. Fixed in Helly's fluid, and stained with Heidenhain's iron-hematoxylin. x 2 000. Chromophobe cell containing nucleus which nodular or droplet-like chromatin granules are distributed irregularly (a), eosinophile cells possessing metachro­ matic (b) and pyknotic nuclei (c), eosinophile cells containing small (d), moder­ ate (e) and abundant cytoplasm (/). FIG. 8. Pars nervosa and pars intermedia of mouse injected with 0.1 R.U. Praehormon for 14 days. Fixed in Helly's fluid, and stained by hematoxylin-eosin stain. x2 000. Parenchymal cells in the pars intermedia containing so-called "nucleus-vacuole" (a) and sinusoidal enlargement and hyperaemia in blood capillaries of the pars nervosa (b). THE NAGOYA jOURNAL OF MEDICAL SCIENCE. VOL. 19 PLATE 11.

f b g

d

1 2 d

c d e 3 4

T. Aoi: Experimental·Histological Studies on the Changes in the Pituitary of the A1ouse Following Administration of Anterior Lobe Extract ( Praehormon). THE NAGOYA JOURNAL OF MEDICAL SCIENCE. VOL. 19 PLATE 12.

5 6

I

d

e

a 7 8

T. Aoi: Experimental-Histological Studies on the Changes in the Pituitary of the Mou~e Following Administration of Anterior Lobe Extract (Praehormon ).