Department of Endocrine Pharmacology, Division of Clinical Research, Department of Experimental Toxicology, Research Laboratories of Schering AG,

Berlin/Bergkamen,

Müllerstrasse 170-178, 1000 Berlin 65, FRG

EFFECT OF PROLACTIN INHIBITORY AGENTS ON THE ECTOPIC ANTERIOR PITUITARY AND THE MAMMARY GLAND IN RATS

By

K.-J. Gr\l=a"\f,R. Horowski

and M. F. El Etreby

ABSTRACT The purpose of the present study was to investigate the biological effectiveness of two highly potent prolactin (PRL) inhibitors, lisuride hydrogen maleate (LMH) and 2-Br-\g=a\-ergocryptine(CB-154), in the absence of hypothalamic factors acting directly at the level of the anterior pituitary. Hypophysectomized female rats bearing 4 transplanted pituitaries beneath the kidney capsules were treated with oestradiol benzoate (OeB) and progesterone (P) with or without simultaneous administration of LHM or CB-154 for 22 days in order stimulate or inhibit lobulo-alveolar growth of the mammary glands. In addition to the investigation of the mammary glands by DNA determination and assessment of the histological pictures, the aim of this study was directed towards the influence of the substances tested at the level of the anterior pituitary remote from the hypothalmus. In this connection the changes in the different cells within the ectopic pituitaries as revealed by immunoenzyme-cytochemical studies were inves-

tigated. The results obtained support the classical view of a neuroendocrine of mammary gland growth and the importance of oestrogens, P and PRL within this system. Both ergot derivatives LHM and CB-154 were able to antagonize the stimulatory effect of OeB combined with P on the mammary gland. With regard to the mechanism of action of LHM and CB-154 it is concluded that both substances act via a direct action on dopaminergic receptors within the ectopic anterior pituitary.

regulation

the phase of the reproductive cycle and the species investigated various hormones have been claimed as being involved directly or indirectly in the development and function of the mammary gland (for review see Lyons et al. 1958; Cowie 8c Tindal 1971). In rats it has been shown that the ovarian steroids progesterone and oestro¬ gens, together with the hypophyseal hormones prolactin (PRL) and growth hormone (GH), are the essential constituents of a hormonal complex necessary for the fully developed mammary gland. This is in agreement with the concept of a functional unit of the ovaries and the pituitary with its neural intercon¬ nections. The hypophysectomized rat bearing ectopic pituitaries proved to be a very useful model for the investigation of this unit. Under these conditions the inhibitory hypothalamic action on prolactin synthesis and release as well as other influences such as that of releasing hormones and stress factors are eliminated. This model thus guarantees rather constantly elevated peripheral PRL levels and, conversely, low or unmeasurable LH, FSH, and GH levels. Because of this we were tempted to investigate the mechanism of action of two highly potent PRL-inhibitors at the level of the pituitaries transplanted to an anatomical site remote from the hypothalamus and also to study its consequences for the mammary gland as the trophic organ for the mammotrophic hormone complex.

Depending

on

MATERIALS AND METHODS Female CFHB rats (supplied by Carworth Europe) weighing 180-200 g were hypo¬ physectomized. To secure PRL-release, the rats received 4 pituitary grafts each transplanted beneath the kidney capsules one to two days later. The pituitary trans¬ plants were taken from immature female rats of the same strain. All surgically treated rats were housed in groups of 4 to 6 animals under natural daylight conditions and fed Herilan Han MR5 fortified diet and water or glucose ad libitum. After a period of involution lasting about 14 days the rats were randomized and split into 3 experimental groups. All rats were treated daily with 5 fig/kg oestradiol benzoate (OeB) together with 50 mg/kg progesterone (P) given subcutaneously for 22 days. Depending on the experimental group, the rats were given additional daily subcutaneous treatment for 22 days of 0.9 (wt/vol) °/o NaCl (experimental group 1), 2 mg/kg CB-154 (experimental group 2), or 0.1 mg/kg lisuride hydrogen maléate1) (LHM) (experimental group 3). At the end of the experiment the following paramètres

investigated: a) Immunoenzyme-cytochemical investigations of the pituitary grafts b) Radioimmunological determination of the serum PRL-concentrations c) Histology of the mammary gland d) DNA determination of the mammary gland.

were

')

synthesized by SPOFA, Prague 268

General experimental procedures OeB and P were dissolved in sesame oil (5tig

or 50 mg/ml), while LHM and CB-154 or 2 "la NaCl given (0.1 mg (wt/vol) mg/ml). In experimental group 1 the rats were killed by decapitation in groups at 11 a.m., 5 p.m. or 9 p.m., while the animals of experimental groups 2 and 3 were all killed at 11 a.m. The blood was collected in centrifuge tubes, allowed to clot at 4°C and centrifuged at 3000 x g for 10 min. The serum was removed and stored at -20°C until assayed for PRL, LH and FSH. The hormones were estimated using the radioimmu¬ noassay materials supplied by the Rat Pituitary Hormone Distribution Program of the National Institute of Arthritis, Metabolism and Digestive Disease (NIAMDD). The assay procedures using the double antibody radioimmunoassay were those recommended by the NIAMDD. The results are expressed in terms of the rat PRL reference standard (NIAMDD RAT PRL RP-1) in ng/ml as the mean (± sem) of all samples/group measured. The grafted pituitaries were removed immediately after decapitation and fixed separately in Bouin's fluid. After embedding in paraffin 3-5 /02 together with 3,3'diaminobenzidine as the first substrate and H202 together with 4-chloro-l-naphtol as a second substrate. The DNA content was measured in one inguinal mammary pad excised from each animal according to the method described by Webb 8c Levy (1955) with only minor modifications. The other inguinal mammary pad was excised from each animal and fixed in Bouin's fluid. For histological examination sections with a mean thickness of 5 ftm were prepared and stained with haematoxylin-eosin. Fig. 1. Ectopic pituitary grafts of the different experimental groups. 1 a—c: Immunoperoxidase staining of PRL cells, yellow-brown stained, using anti-rat-PRL and H202 + 2,3'-diaminobenzidine. 1 d-f: Simultaneous immunoperoxidase staining of PRL cells (brown stained) and GH-cells (greyish-violet stained), using anti-HGH and H202 + 4-chIor-l-naphtol, x 680. Fig. 1 a. Experimental group 1, OeB + P. Note the high proliferative and secretory activity of PRL-cells, which can be recognized by the appearance of mitotic figures (M), enlarged cells with large vesicular nuclei and pale cytoplasmic staining. Experimental

group 2, OeB

of

+

P

+

regression

Fig. 1 b. CB-154. The PRL-cells show (see also under results).

striking signs

of

Fig. 1 c. Experimental group 3, OeB + P + LHM. In comparison with Fig. 3 a, the PRL-cells are less frequent, irregular in form, with signs of vacuolar degenerative changes and clumping of cytoplasm (see also under results).

Fig. 1 d. Experimental group 1, OeB + P. Note the predominance of the PRL-cells (brown stained) in comparison with the GH-cells (greyish violet stained). Fig. 1 e. Experimental group 2, OeB + P + CB-154. Whereas the GH-cells seemed to be only slightly affected, the PRL-cells are clearly reduced with clear signs of regression. Fig. 1 f. Experimental group 3, OeB + P + LHM. In comparison with Fig. 1 d, the PRL-cells occur less frequently, with signs of regression. In contrast to Fig. 1 e the PRL-cells retained their round or polygonal form. 270

271

RESULTS

The histological examination of the pituitary grafts revealed that, in all the different groups, the transplants were well vascularized. With regard to their cellular composition they show marked uniformity within each group. Only a few of the grafts had undergone some scarring with plentiful nuclear pyknosis and no or little evidence for functional cells. The pituitary grafts from the rats treated with OeB together with P (experimental group 1) showed that the PRL-cells are very numerous, highly proliferative with frequent mitosis and secretory activity (see Fig. 1 a and 1 d). As regards cell types other than the PRL-producing cells clear survival of corticotrophic, thyrotrophic, gonadotrophic and somatotrophic cell types could still be detected in most of the pituitaries even 5 weeks after grafting. All those cell types apart from the PRL-cells show signs of involution such as clumping and high vacuolisation of cytoplasm as well as nuclear degeneration. -

-

Fig. 2. Effect of CB-154 and LHM on serum prolactin concentrations in hypophysectomized female rats bearing 4 ectopic pituitaries pre-treated with oestradiol benzoate (OeB) and progesterone (P). 272

The

PRL-producing cells of the rats treated additionally with CB-154 (experimental group 2) displayed striking morphological signs of regression (see Fig. 1 b and 1 e). They are few in number, singly distributed, frequently irregular in form, and they tend to be flattened or stellate in shape. The nuclei appear to be very small and surrounded by a narrow rim of cytoplasm. The severe atrophy, regression and shrinkage of the prolactin cells is usually ac¬ companied by intense homogeneous staining of the cytoplasm, denoting a high hormone content. Comparable to CB-154, LHM (experimental group 3) also led to a marked regression of the stimulatory effect of the concurrent oestrogenprogesterone treatment on the PRL-producing cells (see Fig. 1 c and 1 f). Again there was a clear reduction in the number and size of these cells, which retained their round or polygonal form. This is accompanied by signs of vacuolar degeneration and clumping of cytoplasm. Furthermore, they appear to be either singly distributed or to occur in small groups, and exhibit pale granular staining. The radioimmunological determination of the serum PRL-concentrations parallel the morphological findings within the pituitary grafts. In order to prove a rhythmical release in response to the oestrogen-progesterone treatment, serum PRL-concentrations were investigated at 11 a.m., 5 p.m. and 9 p.m. in experimental group 1, and at 11 a.m. only in experimental group 2 and 3. The serum PRL-concentrations are markedly elevated in response to the con¬ current treatment with OeB and P. There was no rhythmical pattern detectable at the time of investigation (see Fig. 2). The additional treatment with either CB-154 or LMH lowered the radioimmunoassayable PRL-concentrations to nearly control level, as can be measured in hypophysectomized pituitarytransplanted rats without any additional treatment (see Fig. 2). As regards the effect of CB-154 and LHM, there was no significant difference in their PRL-lowering potency, although it must be stated here that LHM proved to be equal in effect to CB-154, even with the very low doses (0.1 mg/kg) used. For control studies, both serum LH and serum FSH proved to be undetectable in all the experimental groups, indicating a complete interruption of the pituitary-hypothalamic interconnections. The histology of the mammary glands of the rats treated with OeB together with P displayed obvious stimulation of lobulo-alveolar growth (see Fig. 3 a). CB-154 as well as LHM markedly inhibited this stimulatory effect (see Fig. 3 b and 3 c). The histological investigations were supplemented by determinations of the DNA content of one inguinal mammary gland pad as a measure of cell num¬ bers or mitotic activity and thus for the extent of proliferation. There was close agreement between the DNA content and the histological findings, thus quantifying the degree of stimulation or inhibition according to the different schemes of treatment. While the combined treatment with OeB and P led to 273 Acta endocr. 85, 2

is

Fig. 3. Histology

of mammary

gland

of the different

274

experimental

groups,

x

16.

Fig. 4.

Effect of CB-154 and lisuride hydrogen maléate (LHM) on DNA content and dry weights of mammary glands in hypophysectomized female rats bearing 4 transplanted pituitaries and treated for 3 weeks with 5 mg/kg oestradiol benzoate together with 50

mg/kg progesterone.

marked increase in the DNA content, this effect is completely antagonized by the concurrent treatment with either CB-154 or LHM (see Fig. 4). As usual, the dry weights of the mammary pads investigated run exactly parallel to the DNA concentrations. a

Fig. 3 a.

Experimental

group 1, OeB

+

P. Note the stimulated lobulo-alveolar

growth.

Fig. 3 b. Experimental Fig. 3 a, the

group 2, OeB

+

lobulo-alveolar

P

+

CB-154. In comparison with is completely inhibited.

growth

Fig. 3 c. Experimental group 3, OeB + P + LHM. As in Fig. 3 b, no lobulo-alveolar growth can be seen. 275 18*

DISCUSSION

The present work supports the classical view of a neuroendocrine regulation of mammary gland growth and the importance of oestrogens, P and PRL within this system (for review see Cowie 8c Tindal 1971). As regards the role of the pituitary, the use of CB-154 and LHM, two highly potent PRL-inhibitors (del Pozo 8c Fliickiger 1973; Graf et al. 1976) has substantiated the important role played by PRL within this regulatory unit. Since the predominant role of PRL within the mammotrophic hormone complex has been generally accep¬ ted, the main aim of our present studies has been directed to the mechanism of action of LHM and CB-154 at the level of the ectopic pituitary and its consequences in respect of mammary gland growth. Our results clearly confirm that both ergot derivatives are acting directly at the level of the anterior pituitary (del Pozo 8c Fliickiger 1973; Horowski 8c Graf, in press), al¬ though additional central actions are not ruled out. Furthermore, the im¬ munoenzyme-cytochemical data on the various cell types investigated point to a very selective influence of CB-154 and LHM at the PRL-producing cells while, in consequence of their isolation from the stimulatory influences of the hypothalamus, the other cell types seemed to be generally regressed but still detectable even 5 weeks after the transplantation procedures. Neither CB-154 nor LHM proved to have any significant effect on these cells. While it is known that oestrogens and synthetic progestogens with oestrogenic activity led directly at the level of the anterior pituitary to hyperplasia and hypertrophy of prolactin cells (Baker et al. 1973; Gersten Se Baker 1970), the present data add the morphological proof of the stimulatory action of OeB together with P and, concersely, the inhibitory effect of CB-154 and LHM on the PRLproducing cells within the ectopic pituitary. This is in good agreement with and complements previous knowledge, mainly gained from in vitro studies, measurements of PRL-content and PRL-release from incubated pituitary half after various treatments, including ergot derivatives, and reflects a more dy¬ namic picture in respect of their influence on PRL-synthesis and/or secretion (MacLeod 1976) (MacLeod 8c Lehmeyer 1974). Our immunocytochemical data together with the corresponding results regarding the radioimmunoassayable serum PRL-concentrations confirm that CB-154 and LHM act by inhibiting the proliferative potency of PRL-cells as well as PRL-synthesis and/or release. This correlates with studies which state that CB-154 inhibits proliferation of oestrogen-stimulated PRL-producing cells but raises the PRL-concentrations within the pituitary itself (Davies et al. 1974; Lloyd et al. 1975). With regard to the effect of oestrogen on PRL-cells, these results in turn have been inter¬ preted as meaning that PRL-cells cannot divide under the influence of oe¬ strogens unless they are free to release PRL, thus suggesting an intracellular negative feedback mechanism (Lloyd et al. 1975). Based on this hypothesis. 276

the comparison of the effects of LHM and CB-154 on the PRL-cells in our study suggests that LHM acts by inhibition of PRL-synthesis and release, as might be reflected by the morphological appearance and the light and granular staining of these cells, indicating a rather low hormone content. In contrast, CB-154 seems to affect mainly PRL-release only (del Pozo 8c Fliickiger 1973), a finding which is substantiated in our study by the severe atrophy and shrinkage of PRL-producing cells, which are dark and homogeneously stained as a possible index of a relatively high hormone content. However, these ob¬ servations may also be explained by the different doses used and thus as a quantitive phenomenon. The described effects at the level of the prolactin producing cells together with the known serum PRL-lowering potency of both LHM and CB-154 is consistent with the now generally accepted concept that dopamine has a predominant inhibitory influence on the regulation of PRLsynthesis and/or secretion (MacLeod 8c Lehmeyer 1974; Horowski 8c Graf, in press). In the case of the two PRL-inhibitors investigated the PRLlowering effect seems to be directly related to their known dopaminergic activities (Corrodi et al. 1973; Horowski 8c Wachtel 1976). In this connection our results support the hypothesis that dopaminergic receptors affecting PRLsynthesis and/or secretion are located within the pituitary itself. With regard to the mammary gland, the present data provide additional evidence for the biological effectiveness of LHM at suppressing lobulo-alveolar growth, most likely as a consequence of its PRL-inhibitory activity. These results are in line with the inhibitory effect of LHM on lactation (Ausková et al. 1974), thus indicating a close parallelism to the known biological properties of CB-154.

ACKNOWLEDGMENTS The authors wish to thank Dr. Nishino for the DNA determination and Dr. R. von Berswordt-Wallrabe for the performance of the hypophysectomies, including the trans-

platation procedures. Furthermore, the skilful technical assistance of Mrs. P. Kuhlmann, Miss G. Borowicz, Mrs. A. Esch and Miss U. Tüshaus is greatly appreciated. We are indebted to the NIAMDD Rat Pituitary Hormone Program and to the NIH for supplying the material required for the radioimmunoassays and the immunoenzyme-

cytochemical investigations. We are grateful to Dr. B. L. Baker for anti-ACTH. CB-154 was kindly

for the

gift

provided by Sandoz AG,

of anti-HGH and to Dr. P. K. Nakane

Switzerland.

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