Combined Effects of Testosterone and Estradici on Rat Ventral Prostrate in Organ Culture1

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[CANCER RESEARCH 38. 4126-4134, November 1978] 0008-5472/78/0038-OOOOS02.00 Combined Effects of Testosterone and Estradici on Rat Ventral Prostrate in Organ Culture1

ThÃ©rÃ¨seFeyel-Cabanes, Jean Secchi, Paul Rebel, and Etienne-Emile Baulieu UnitÃ©deRecherche sur le MÃ©tabolismeMolÃ©culaire,et la Physio-Pathologie des StÃ©roides,de l'Institut National de la SantÃ©etde la Recherche MÃ©dicale (U 33 Inserm) and ER 125 du CNRS, HÃ´pitalde BicÃ©tre,78 rue du GÃ©nÃ©ralLedere.94270 BicÃªtre, France Â¡T.F-C., P. R., E-E. B.Â¡,and Roussel-Uclaf, 102 route de Noisy, b.p. n. 9, 93230 Romainvil/e, France [J. S.]

Abstract increases, the present studies may prove to be of interest for the study of benign prostatic hypertrophy (14). Explants of ventral prostate from normal 7- to 8-week- old Wistar rats were cultured for 144 hr Â¡nthepresence of testosterone (1 nMto 1 /UM)and/or estradici (1 nMto 1 Â¿prostates from different animals Chemicals. Estradiol, testosterone, and diethylstilbestrol were performed at each hormone concentration. Mor were obtained from Roussel-Uclaf, Romainville, France, phology was assessed by light and electron microscopy. and tamoxifen was from International Chemical Industries, In the absence of hormone, the prostatic epithelium Macclesfield, England. Medium 199 was from Flow Labora regressed and the nonepithelial components increased. tories, Ltd., Irvine, Scotland. The latter included the perialveolar sheath of smooth Animals. Male Wistar rats were obtained from Iffa Credo, muscular cells and the interstitial stroma with many fibro- St. Germain sur l'Arbresle, France, and sacrificed at 7 to 8 blasts, active macrophages, and a thick network of colla weeks of age. gen. The presence of estradiol (1 nM to 10 /Â¿M)didnot Organ Culture. Expiants of ventral prostate were cultured change this picture, whereas testosterone (1 to 100 nM) in serum-free medium as previously described (2). After a maintained the epithelial cells and prevented the increase 24-hr preincubation in hormone-free medium (to eliminate of the perialveolar sheath and interstitial stroma so that any interference by endogenous hormones), the expiants the histolÃ³gica! picture was like that observed in the were transferred into medium containing estradiol and/or young intact adult animal. Any concentration of estradiol testosterone. Hormones were added to the medium Â¡n (1 RMto 1 IJ>M)associated with a physiological concentra propylene glycol. Control cultures in hormone-free medium tion of testosterone (1 to 4 nM) counteracted the andro- received an equal volume of propylene glycol. After 72 hr gen-induced inhibition of the stroma, so that both epithe all expiants were transferred into similar, fresh media for a lium and stroma were simultaneously developed. How further 72 hr. For each treatment examined a minimum of ever, when combined with supraphysiological concentra 10 expiants from different animals were cultured. tions of testosterone (10 to 100 nM), estradiol was no Light Microscopy. Expiants were fixed in Bouin's longer effective and the stroma remained minimal. The aqueous fixative, cut into 5-/um sections, and stained with distribution of mucopolysaccharides and alkaline phos- either hematoxylin-eosin or PapamiltiadÃ¨s trichrome (23). phatase correlated well with the morphological observa Electron Microscopy. Expiants were fixed in 1% osmic tions. acid in Millonig's buffer for 1 hr atOÂ°,dehydrated in alcohol, The ratio of estradiol to testosterone is therefore insuf and embedded in Epon. Ultrathin sections were treated ficient to explain whether or not interstitial tissue will with 5% uranyl acetate and lead nitrate and were examined develop and a critical factor appears to be the absolute with a Siemens Elmiskop 101 electron microscope. concentration of testosterone. Preliminary observations Expiants fixed in Bouin's aqueous fixative were cut and have indicated that diethylstilbestrol does not mimic es stained with Periodic acid-Schiff to reveal the mucopoly tradiol action and that tamoxifen is neither an agonist nor saccharides. For evaluation of alkaline phosphatase ac an antagonist of the estradiol effect. Accordingly, the cording to Gomori (12), expiants were fixed in 80Â°alcohol at estradiol effect obtained at physiological concentrations 0Â°. of testosterone does not seem to be mediated through active participation of a classical estradiol receptor. Results Different concentrations of testosterone and/or estradiol Introduction were tested (Table 1). Light and electron microscopy pic As part of our studies on hormone effects on rat ventral tures were obtained under 6 typical hormonal situations: (a) prostate in organ culture (2), we have been interested in the the situation in the young adult intact animal, where pre activities of estrogens in conjunction with androgen action. sumably testosterone plays the major role (controls); (ft) Some observations in in vitro systems have already been culture with no hormone; (c) culture with testosterone published (10, 18, 19). Since it is known that in the aging alone; (d) culture with estradiol alone; (e) culture with the human the ratio of circulating estradiol to testosterone association of physiological concentrations of testosterone and estradiol; or (f) culture with the association of supra-

1 Presented at the John E. Fogarty International Center Conference on physiological concentrations of testosterone and estradiol Hormonesand Cancer. March 29 to 31. 1978. Bethesda. Md. (Figs. 1 and 2).

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Table 1 sponded to the presence of estradiol. In all cases, periodic Effects of testosterone and/or estradiol on rat ventral prostate acid-Schiff and alkaline phosphatase reactions were mainly epithelium and stroma cells in organ culture found in the stimulated cells. Symbols apply to the images obtained by Jight and electron Preliminary investigations were made with diethylstilbes- microscopy and to the distribution of mucopolysaccharides and trol, a nonsteroidal estrogen (0.1 nM to 1 /Â¿M).aloneor with alkaline phosphatase, as seen in Figs. 1, 2, and 3 and in the text. 4 nM of testosterone. The effect of diethylstilbestrol alone Epithe Stroma'' lium" did not differ from that of estradiol alone, but when diethyl stilbestrol was combined with a physiological concentra Intact animal tion of testosterone there was no antagonistic effect of the androgen-dependent inhibition of the stromal development, Culture No hormone thus showing no estradiol-like activity in the prostate. Testosterone (1-100 nM) Tamoxifen, an antiestrogen with mixed estrogenic ago Estradiol (1 nw-1 /*M) nist and antagonist activity on the rat uterus (13) and purely Testosterone (1 nM) + estradiol (10 nw); Testosterone (4 nM) + estradiol antagonist activity in the chick oviduct (27), was used alone (1 nM-1 /xM) (1 to 100 nM), in the presence of testosterone (1 nM) or of Testosterone (10 nM) + estradiol (1-10 estradiol (1 to 10 nM), and in the presence of both testoster nw); testosterone (100 nw) + one (4 nM) and estradiol (1 to 10 nM). Tamoxifen showed no estradiol (10-100 nM); testosterone effect on its own and did not modify the effects of testoster (1 Â¿IM)+estradiol (1 Â¡J.M) " + + + + , maintained as in intact animals; Â¿,regressed. one and estradiol when used separately or in combination. * Â±,poorly developed as in intact animals; + + + + , developed as These last results indicate a lack of antiestradiol activity in in the absence of hormones. this system. At all concentrations of testosterone >1 nM, the prostatic Discussion expiants were maintained in a state comparable to that The effects of estradiol. alone or associated with testos observed before culture, confirming preceding observa terone, have been studied in seminal vesicles and prostate tions (2). The glandular epithelium was well developed, in several species. These in vivo observations have been with tall columnar cells showing microvilli and signs of difficult to interpret due to complex indirect hormonal active secretory activity (Fig. 3). The perialveolar sheath changes. However, such hormone treatments of castrated was composed of 1 or 2 layers of smooth muscular cells or prepubertal animals (4, 6, 7,11,15,17, 20, 22, 24, 25, 28, and of fibroblasts (9). The interalveolar stroma was reduced 30) caused an increase of the muscular and stromal com to a few dispersed fibroblasts and macrophages. Periodic ponents of the accessory sexual glands. The present in acid-Schiff and alkaline phosphatase staining predomi vitro experiments essentially confirm the observations in nated in the epithelial cells (8). vivo. The use of organ culture in a completely hormone- In the absence of testosterone, the alveolar diameter was deprived medium allowed it to operate at known concentra reduced, the epithelium was atrophied, and its cells were tions of both hormones and to exclude extra prostatic flattened, with the disappearance of secretory granules and changes. microvilli. In addition, they exhibited dense bodies. In The effects of testosterone on ventral prostate can be contrast, the perialveolar sheath was thickened; retracted reproduced partially in an in vitro culture system (2). In the muscular cells showed 'holly-leaf" spines and signs of absence of hormone, the regression observed is similar to micropinocytosis. Moreover, the stroma was well devel that after castration. The mechanism for the striking in oped, with many fibroblasts and active macrophages readily crease of smooth muscle cells, fibroblasts, and other visible together with a dense network of collagen fibers stroma cells in culture without testosterone, whether estra (Fig. 2b). Periodic acid-Schiff and alkaline phosphatase diol is present or not, is unknown. Testosterone can appar stains were again predominant in active cells, this time in ently repress this increase when present at physiological the stroma. concentrations but cannot do so when estradiol is present Regardless of the concentration used, estradiol alone did at the same time. This antitestosterone effect of estradiol is not alter the morphology observed without hormone. observed only on the stroma and not on the epithelial cells When estradiol (>0.1 nM) was added to physiological which remain fully stimulated. However, at supraphysio concentrations of testosterone, i.e., 1 or 4 nM, the epithe logical concentrations, testosterone overrides the antago lium was well developed, maybe even a little more stimu nistic effect of any concentration of estradiol, and the lated than with testosterone alone. However, the interstitial stroma remains repressed by the androgen. These results tissue was also developed, almost as much as in the suggest a greater sensitivity of the stroma cells than of absence of hormones or in the presence of estradiol alone. epithelial cells to the antiandrogenic effects of estradiol. All criteria observed separately in androgen-stimulated epi In examining the literature, we feel that to date no thelial cells and in no-hormone or estradiol-exposed stroma estrogen receptor has been unequivocally identified in rat cells were observed simultaneously when testosterone (1 to ventral prostate, and thus far our own experiments certainly 4 nw) and estradiol were present together (Fig. 4). do not demonstrate such an estradiol receptor (J. P. However, when estradiol (1 nM to 1 /J.M)was added to Blondeau and P. Robel, unpublished data). However, an supraphysiological concentrations of testosterone (10 nM estrogen receptor has been found in sex accessory glands and more), the latter still provoked its typical androgenic from the guinea pig (3). Even if there is an estrogen recep action on the epithelium, but the stroma no longer re tor, it does not show the usual hormone specificity as in

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Table 2 Robel, P. Morphological and Biochemical Parameters of Androgen Affinity of natural and synthetic hormones for receptors Effects on Rat Ventral Prostate in Organ Culture. Vitamins Hormones, 33. 1-38. 1975. Composite schematic table summarizing the affinity of different 3 Belis. J. A., Blume, C. D..Cricco, R. P., Dieringer, C. S., and Mawhinney. compounds for known androgen and estrogen receptors (1, 5). M. G. Relationship of Estrogen Receptors to Fibromuscular Hyperplasia in Male Sex Accessory Organs. The Endocrine Society, 58th Annual Affinity for Meeting, June 23 to 25, 1976, San Francisco, p. 351, Abstract 589. 4 Bern. H. A. Estrogen and Alkaline Phosphatase Activity in the Genital Estrogen Androgen Tract of the Male Mouse. Endocrinology, 48: 25-33. 1951 receptor receptor 5. Blondeau. J P.. Corpechot, C.. Le Goascogne. C., Baulieu, E. E., and Robel. P. Androgen Receptors in the Rat Ventral Prostate and Their Estradici Hormonal Control. Vitamins Hormones. 33. 319-345, 1975. Diethylstilbestrol 6. Courrier, R., and Gros. G. Action de la Foliculine chez le Singe MÃ¢le Tamoxifen ImpubÃ¨re. Modification des Annexes. Compt. Rend. Soc. Biol.. 118: Androstanolone-testosterone 686-689. 1934 7. Emmens. C. W.. and Parker, A. S. Effect of Exogenous Estrogens on the 0.1 RM; + + , Kd Â»10 nM; Â±.K,, Â»1 MM. Male Mammal. Vitamins Hormones, 5. 233-272, 1947. 8 Feyel-Cabanes. T.. Robel, P.. and Baulieu. E. E. Effects Conjoints de la other estrogen target cells if we judge by the effects of Testosterone et de I Oestradiol sur le Lobe Ventral de la Prostate de Rat estrogens here reported. In fact, diethylstilbestrol, which en Culture Organotypique. Compt. Rend. Ser. D, 285: 1119-1122, 1977. 9. Flickinger. C. J. The Fine Structure of the Interstitial Tissue of Rat displays estrogenic activity and high binding affinity for all Prostate. Am. J. Anat., 134: 107-126, 1972. known estrogen receptors (Table 2) (1), could not replace 10 Franks. L. M. The Effects of Age on the Structure and Response to Oestrogens and Testosterone of the Mouse Prostate in Organ Cultures. estradiol, at least in the range of concentrations that we Brit. J. Cancer, 13: 59-68, 1959. tested. Similarly, tamoxifen, which is an antiestrogen act 11. Freud, J. Conditions of Hypertrophy of the Seminal Vesicles in Rats. ing at the estrogen receptor level in several systems (13,16, Biochem.J., 27. 1438-1450,1933. 12. Gomori, G. The Study of Enzymes in Tissue Sections Am. J. Clin. 17), did not antagonize estradiol action (neither did it dis Pathol.. 16: 347-352. 1946. play any estradiol-like activity). Thus the estrogen specific 13. Harper, M. J. K., and Walpole. A L A New Derivative of Triphenyleth- ity of the effect does not correspond to the binding specific ylene: Effect on Implantation and Mode of Action in Rats. J. Reprod. ity of "ordinary" estrogen receptors. We suggest the possi Fertility. 13: 101-119, 1967. 14. Hechter, 0. Reflections on the NIH Workshop on Bph. In: J.T. Grayhack. bility that estradiol acts via the androgen receptor since it J. D. Wilson, and M J. Scherbenske (eds.), Benign Prostatic Hyperpla sia. DHEW Publication No. (NIH) 76-1113, pp. 269-281. 1976. binds to this receptor with high affinity (5), while both 15. Huggins. C.. and Johnson Clark, P. Quantitative Studies of Prostatic diethylstilbestrol and tamoxifen show only negligible affin Secretion. II. The Effect of Castration and of Estrogen Injection on the Normal and on the Hyperplastic Prostate Glands of Dogs. J. Exptl Med.. ity (Table 2). If the estradiol effect takes place at the andro 72: 747-762, 1940. gen receptor level, one has still to decide whether it con 16. Jordan, V. C.. Dix, C. J., Rowsby, L., and Prestwich, G. Studies on the cerns an androgen receptor in epithelial and/or stroma Mechanism of Action of the Nonsteroidal Antioestrogen Tamoxifen (ICI 46. 474) in the Rat. Mol. Cellular Endocrinol., 7: 177-192. 1977. cells. Autoradiographic experiments have shown that 17. Korenchewsky, V.. and Dennison, M. Histological Changes in the androgen receptors are located mainly if not exclusively in Organs of Rats Injected with Destrone Alone or Simultaneously with epithelial cells of rat ventral prostate (5, 26, 29). Therefore, Destrone and Testicular Hormone. J. Pathol. Bacteriol.. 41: 323-337, 1935. it is not excluded that the inhibitory effect of testosterone 18. Lasnitzki, I. The Effect of Hydrocortisone on the Ventral and Anterior on interstitial stroma could be indirect, as, consequently, Prostate Gland of the Rat Grown in Culture. J. Endocrinol.. 30: 225-233. could estradiol activity. 1964. 19. Lasnitzki, I. The Effect of Hormones on Rat Prostatic Epithelium in Estrogens are known to inhibit testosterone 5Â«-reduc- Organ Culture. In: M. Goland (Ã©d.).Normal and Abnormal Growth of the tase. A selective inhibition by natural estrogens such as Prostate, pp. 29-54. Springfield. III.: Charles C Thomas. Publisher, 1975. 20. Neumann, F., Richter, K. D., and Penge, T. Animal Models in the Study estradiol. compared to synthetic diethylstilbestrol. could of Antiprostatic Drugs. Vitamins Hormones. 33: 103-135. 1975. explain the difference observed between the 2 estrogens. 21. Nozu, K., and Tamaoki, B. I. Characteristics of the Nuclear and Micro- However, both compounds have been shown to give similar somal Steroid A'-SnHydrogenase of the Rat Prostate. Acta Endocrinol.. 76. 608-624, 1974. inhibition of prostatic 5Â«-reductase (21). 22. Overholser. M., and Nelson. W. The Effect of Estrin and Male Hormone Until now the rat prostate has not been considered Injected Separately and Simultaneously upon the Smooth Muscle and suitable for the study of human prostatic disease. In partic Epithelium of the Seminal Vesicle in the Albino Rat. Anat. Record, 62: 247-267, 1935. ular, no morphological abnormalities, spontaneous or ex 23. Papamiltiades, M. Une MÃ©thode de Coloration Rapide de l'Hypophyse perimentally induced, have been observed that resemble par l'HÃ©matoxyline au Lugol. Compt. Rend. Assoc. Anat., 92: 1151- 1155.1955 the human benign prostatic hypertrophy. It is therefore 24. Price, D., and Williams-Ashman, H. G. The Accessory Reproductive extremely interesting that the hormones and organ culture Glands of Mammals. In: W. C Young, (ed.), Sex and Internal Secretions, technique used here have permitted us to obtain expiants Vol. 1, p. 366. Baltimore: The Williams and WilkinsCo.. 1971. 25. Salander, H.. and Tisell. L. E. Effects of Megestrol on Oestradiol Induced in which the epithelial and stroma cells are simultaneously Growth on the Prostatic Lobes and the Seminal Vesicles in Castrated developed as in this disease. Rats Acta Endocrinol., 82: 213-224. 1976. 26. Sar. M., Liao. S.. and Stumpf. W. E. Nuclear Concentration of Andro- gens in Rat Seminal Vesicles and Prostate Demonstrated by Dry-Mount Acknowledgments Autoradiography. Endocrinology, 86 1008-1011, 1970. 27. Sutherland, R.. Mester. J.. and Baulieu. E. E. Tamoxifen Is a Potent We thank Marie-ThÃ©rÃ¨se Picard-Groyer and Alberte Holbach for their very "Pure" Anti-oestrogen in Chick Oviduct. Nature, 267: 434-435. 1977. valuable help. Heather Mullis for the preparation of the manuscript, and Roussel-Uclaf for partial support. 28. Tisell, L. E. The Growth of the Ventral Prostate, the Dorsolateral Prostate, the Coagulating Glands and the Seminal Vesicles in Castrated Adrenalectomized Rats Injected with Oestradiol and/or Cortisone. Acta References Endocrinol.. 68: 485-501. 1971. 1. Baulieu, E. E., Alger. M . Best-Belpomme. M., Corvol, P., Courvalin, J. 29 Tveter. K. J , and Attramadal. A. Autoradiographic Localization of C., Mester. J.. Milgrom, E., Robel. P.. Rochefort, H. and de Catalogne, Androgen in the Rat Ventral Prostate. Endocrinology. 85: 350-354,1969. D. Steroid Hormone Receptors. Vitamins Hormones. 33: 649-731. 1975 30. Walsh. P. C.. and Wilson. J. D. The Induction of Prostatic Hypertrophy in 2. Baulieu. E E.. Le Goascogne. C.. Groyer, A.. Feyel-Cabanes, T . and the Dog with Androstanediol. J. Clin. Invest.. 57: 1093-1097. 1976.

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Fig. 1. Histology of 7-day cultures, a, without hormone; t>, 10 nM estradici; c, 4 nw testosterone;

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Fig. 2. Electron microscopy. Histology.a. intact animal, x 3.650. b to f, 7-day cultures.Ã¹, without hormone, x 4,200.c. 1 nw testosterone, x 3,350.ci. 10 nM estradici, x 4.200. e, 4 nM testosterone with 10 nM estradici, x 3,350. t. 10 nw testosterone with 10 nw estradici, x 3.500. E, epithelium; mv, microvilli; rer, rough endoplasmic reticulum;g. Golgi apparatus: n. nucleus; M. smooth muscle cell; ML. muscular layer (sheath); F. fibroblast; C. collagen;^, dense body; BL. basal lamina; Ma. macrophage; s, secretory granule; L. lumen;C7, connective tissue (stroma);m, mitochondrion.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1978 American Association for Cancer Research. Combined Effects of Testosterone and Estradiol on Rat Ventral Prostrate in Organ Culture

Therese Feyel-Cabanes, Jean Secchi, Paul Robel, et al.

Cancer Res 1978;38:4126-4134.

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