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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 ThérèseFeyel-Cabanes, Jean Secchi, Paul Rebel, and Etienne-Emile Baulieu UnitédeRecherche sur le MétabolismeMoléculaire,et la Physio-Pathologie des Stéroides,de l'Institut National de la Santéetde la Recherche Médicale (U 33 Inserm) and ER 125 du CNRS, Hôpitalde Bicétre,78 rue du GénéralLedere.94270 Bicêtre, France ¡T.F-C., P. R., E-E. B.¡,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 ¡nthepresence of testosterone (1 nMto 1 /UM)and/or estradici (1 nMto 1 ¿<M).Materials and Methods Several experiments with 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 /¿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 histológica! 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 ¡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 Papamiltiadès 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 atO°,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 80°alcohol at estradiol effect obtained at physiological concentrations 0°. 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). 4126 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1978 American Association for Cancer Research. Testosterone and Estradiol Effects on Rat Ventral Prostate 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 /¿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 ¿IM)+estradiol (1 ¡J.M) " + + + + , maintained as in intact animals; ¿,regressed. one and estradiol when used separately or in combination. * ±,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.

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