Mesenchyme-Mediated Effect of Testosterone on Embryonic Mammary Epithelium1

[CANCER RESEARCH 38, 4066-4070. November 1978) 0008-5472/78/0038-OOOOS02.00 Mesenchyme-mediated Effect of Testosterone on Embryonic Mammary Epithelium1

Heidelinde DÃ¼rnberger,Barbara Heuberger, Peter Schwartz,2 Gertraud Wasner, and Klaus Kratochwil

Institut fÃ¼rMolekularbiologie der Ã¶sterreichischen Akademie der Wissenchaften, Billrothstr. 11, A-5020 Salzburg, Austria

Abstract possible endocrine therapy of breast cancer. The embryonic mammary rudiment of the mouse re Extensive information is available on intracellular events that take place after steroid application. The action of the sponds to testosterone with the formation of a condensa tion of mesenchymal cells around the gland bud and hormone is mediated by specific cytoplasmic receptors and involves the translocation of the hormone:receptor complex subsequent necrosis of the gland epithelium. Experimen into the nucleus with subsequent modification of transcrip- tal combinations of epithelium and mesenchyme of this tional activity (7). In our laboratory we are concerned with rudiment, by taking advantage of the androgen-insensitive mutant (Tfm, "testicular feminization"), have shown the intercellular processes that occur within the mammary gland in response to androgenic hormones. The model that the hormone acts directly on the mesenchyme, its system is the embryonic rudiment of the gland in the effect on the epithelium being indirect and mediated by mouse, which has long been known to develop differently the surrounding mesenchyme. Only the mesenchymal in female and male fetuses (22, 25). Experiments in vivo cells at the epithelial surface are capable of initiating this have implied that fetal testicular hormones are the causative reaction, and there is no oriented migration of specialized agents for this sexual dimorphism (20, 21), and we have mesenchymal cells to the gland bud in response to the shown in organ cultures of mammary rudiments that the hormone. The mesenchyme forms this condensation only gland responds directly to androgenic hormones with high around mammary epithelium; in experimental association specificity (9, 10). the epithelia of the embryonic lung, pancreas, or salivary The androgen-induced processes in the mammary gland gland remain unaffected. The experiments suggest fur ther that association of mammary epithelium with "mam take place on Day 14 of gestation, i.e., at a time when the mary" mesenchyme must exist for some time before rudiment consists only of a small epithelial bud attached to the epidermis by a somewhat thinner "stalk." The first sign hormone action to allow a response. of the reaction is the appearance of a conspicuous conden The finding that the destructive action of testosterone sation of mesenchymal cells around the epithelial bud. As on mammary epithelium is mediated by the surrounding this condensation progresses the stalk of the epithelial mesenchyme and requires organ-specific tissue interac rudiment becomes stretched and finally ruptures, separat tion is discussed in regard to its possible significance for ing the mammary epithelium from the epidermis. This an androgen therapy of metastatic breast cancer (i.e., detached piece of gland epithelium, as well as the portion mammary epithelium in nonmammary mesenchyme). of the stalk connected to the epidermis, then undergoes massive necrosis (22). A more detailed description of the Introduction cellular events in response to androgenic hormones will be given elsewhere.3-4 The presence of receptors for androgenic hormones has From histological observations it is obvious that both been demonstrated in samples of human breast cancer (4, tissues, gland epithelium and surrounding mesenchyme, 14, 17) and in mammary carcinoma cell lines of human are involved in the androgen-induced reaction (Fig. 1). (MCF-7; Refs. 6 and 13) and mouse (Shionogi 115; Refs. 2 However, the fact that testosterone was shown to act and 23) origin. Responsiveness to androgens was found in directly on the rudiment (in culture) does not necessarily some cases of human breast cancer (1), in human (13) and imply that it also acts directly on both of its tissues. The mouse (2, 23) cell lines, as well as in dimethylbenzanthra- cene-induced mammary carcinomas of the rat (19, 24). gland could only have one target tissue for the hormone, which then influences the other component through proc Whereas the response of the rat tissue involved regression esses referred to as "tissue interaction." The availability of of the tumor, the mouse and human cell lines were found to an androgen-insensitive mutant of the mouse [Tfm for be stimulated by and even dependent on androgens for "testicular feminization" (16)] makes it possible to identify their growth and maintenance in culture. the tissue responding to the hormone. Since androgenic hormones have been used with some success in the treatment of human breast cancer (1), an Materials and Methods understanding of the mechanism of action of androgens on Normal mouse embryos were hybrids of BALB/c x C3Hf mammary tissue would appear to be highly relevant for a matings. The propagation of the X-chromosome carrying

1 Presented at the John E. Fogarty International Center Conference on 3 P. Schwartz. Die Entwicklung der embryonalen MilchdrÃ¼senanlageder Hormones and Cancer, March 29 to 31, 1978, Bethesda. Md. Supported by Maus, eine elektronenmikroskopische Untersuchung speziell der androgen- National Cancer Institute Contract N01-CB-33883. bedingten RÃ¼ckbildungder Drusenanlage mannlicher Embryonen, submit 2 Present address: Anatomisches Institut der UniversitÃ¤tGÃ¶ttingen,West ted for publication. Germany. 4 P. Schwartz, and K. Kratochwil, Manuscript in preparation.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Tissue Interaction in Androgen Response of Mammary Gland the Tfm mutation in a separate mouse colony (derived from Table 1 3 pairs donated by Mary F. Lyon) has been described (12). The androgen response of 4 types of epitheliomesenchymal Only X'''""/Y hemizygous embryos are androgen-insensitive, combinations and these were distinguished from normal X'/Y males by The X""7Y tissues are androgen-insensitive. All other tissues [X'/Y or wild type - (wt)] are normal with regard to the Tfm locus. testing 3 mammary rudiments of each embryo for androgen Only combination type Â¿,containing androgen-insensitive mesen sensitivity. X'^/X" heterozygous female embryos (derived chyme, did not yield a single responsive gland. from the same carrier females as X'""/Y embryos) could be Androgen response observed distinguished from X /X' littermates by the incomplete Combination type a.X7'"'7Y-epithelium 33/49" response of their mammary glands to testosterone. wt-mesenchyme Dating of embryos and the organ culture of mammary b. wt-epithelium 0/49 rudiments has previously been described in detail (8), as X 'â€¢Â»'/Y-mesenchyme well as the androgen response of intact glands in vitro. For c. X'/Y-epithelium 13/21 the recombination technique see Ref. 12. Clean separation wt-mesenchyme" of epithelium from mesenchyme was carried out after 30 d. wt-epithelium 26/30 min incubation in a solution of 2.25% trypsin (1:250) plus X'/Y-mesenchyme 0.75% pancreatin (N. F., both Difco Laboratories, Detroit, " Number of respondings glands/total number of successful Mich.). Mammary epithelia with few adhering mesenchymal combinations. cells were obtained by careful peeling of the skin of 12- or 13-day embryos (without previous enzymatic treatment). chyme and that the genotype of the epithelium is irrelevant. Mesenchyme-free epithelia of 11-day pancreas, 11-day We assume also that, in the intact gland, testosterone acts lung, and 13-day submandibular salivary gland were ob only on the glandular mesenchyme, which then, in a yet tained after a 5- to 10-min incubation in the same trypsin: unknown fashion, causes epithelial necrosis (12). pancreatin solution (5). Origin of the Mesenchymal Cells Involved in the Andro The androgen response of mammary rudiments as well gen Response. Virtually none of the mesenchymal cells as of experimental tissue combinations was observed in exhibiting the condensation characteristic of the early living expiants and further documented in histological sec stages in the androgen response showed thymidine incor tions after glutaraldehyde fixation and embedding in Epon. poration in radioautographs. The mesenchymal condensa tion therefore arises by accumulation of cells rather than by Results localized proliferation. We wondered whether this accumu lation was a strictly local phenomenon or whether some Identification of the Target Tissue for Testosterone. type of specialized mesenchymal cell would migrate from Explanted mammary rudiments (epithelium with surround more distant sites to the gland bud in response to testoster ing mesenchyme) of normal mouse embryos respond to 1 one. nw testosterone in the medium (9). Histologically, this To test the latter possibility, we combined androgen- response in vitro is identical to the appearance of affected sensitive (wild type) glands containing only a thin coat (2 to mammary rudiments in male fetuses. In contrast, glands of 3 cell layers) of equally androgen-sensitive mesenchymal androgen-insensitive (X'""/Y) embryos remain entirely un cells with a large mass of androgen-insensitive (Tfm) mes affected even with 10 Â¿xMtestosterone. enchyme. All 62 of these combinations responded normally Experimental tissue combinations consisted of either (a) to testosterone, indicating that the mesenchymal cells re androgen-insensitive (X""7Y) gland epithelium and wild- quired to initiate the reaction had already been at the type (BALB/c x C3H) mesenchyme or (b) wild-type epithe epithelial surface at the time of combination (12- to 13-day lium and androgen-insensitive mesenchyme. Since X'""/Y stage). On the other hand, an equally thin coat of androgen- embryos could not be distinguished from their X /Y litter- insensitive mesenchymal cells around the gland epithelium mates at the stage of tissue separation and combination, (of the same developmental stage) prevented the androgen we also prepared combinations of (c) XVY epithelium with reaction in all 82 glands associated with a large mass of BALB x C3H (wild-type) mesenchyme and (d) wild-type normal, androgen-sensitive mesenchyme. This result also epithelium with X'/Y mesenchyme. tends to exclude oriented migration of distant mesenchymal After 3 days in culture with medium containing 0.1 /Â¿M cells to the mammary rudiment as the source of the mes testosterone, we recovered 49 glands of the combination enchymal condensation. It appears that the mesenchymal type a, 33 of which showed a typical androgen response reaction can only be initiated at the epitheliomesenchymal despite the fact that the gland epithelium was from an interface. androgen-insensitive embryo. In contrast, under the same From the previous experiments it was not possible to conditions all 49 glands of the combination type b, contain deduce whether the entire mesenchymal condensation ing androgen-insensitive mesenchyme, remained unaf formed in the first type of combination was composed of fected by the hormone. Both types of the combination androgen-sensitive cells or whether only a thin layer of involving tissues of X'/Y embryos yielded a reasonable such mesenchyme at the epithelial surface is required to percentage of responding glands, thus ruling out strain initiate the reaction, which then spreads into more distant differences independent of the Tfm locus as the reason for mesenchyme, irrespective of its capacity to respond to the the failure of combination b (Table 1). hormone. A close look at the response of explanted glands This result shows that the response of an experimental taken from X'""/X' heterozygous female embryos provided tissue combination requires androgen-sensitive mesen- information pertinent to this problem.

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Due to the inactivation of 1 X-chromosome at an early would allow us to distinguish between these alternatives. embryonic stage, each somatic cell of a female mammal Rather surprisingly, 14-day mesenchyme (i.e., of the re contains only 1 functional X-chromosome (15). Inactivation sponsive stage) did not exhibit an androgen-induced reac occurs at random; but, after this occurs, all descendants of tion around mammary epithelium of any stage, including a cell have the same active X-chromosome. An X-chromo- 14-day epithelium. Conversely, 14-day mammary epithelium some-heterozygous female therefore is a mosaic composed did not elicit a testosterone response in mesenchyme of any of cell clones with different phenotypic properties specified stage between 11 and 15 days. Such a response was only by genes located on the X-chromosome. Consequently, an seen when neither epithelium nor mesenchyme was older Xr""/X' animal consists of cones fully responsive to testos than 12 days at the time of experimental association. Even in "homochronic" combinations, 12-day epithelium associ terone and of clones entirely unresponsive to the same hormone (3, 18). In the histological sections of X'""/X+ ated with 12-day mesenchyme gave a response, whereas glands, we found heterogeneity within the mesenchyme combinations of 13- and 14-day tissues yielded mostly nega after testosterone application: clusters of condensed cells tive results. next to apparently unaffected, loose mesenchyme (Fig. 2). Heterochronic combinations of 11-day epithelium with We assume that the cell condensations, which are always 12-day mesenchyme responded to testosterone after 2 days in contact with mammary epithelium, represent clones of in vitro, whereas the reciprocal association of 12-day epi mesenchymal cells expressing the X-chromosome, thelium with 11-day mesenchyme did so only after 3 days in whereas unaffected cells would be of Tfm phenotype. The culture. It appears therefore that the developmental stage presence of such unaffected mesenchymal cells at the of the mesenchyme is decisive for the earliest possible epithelial periphery and next to condensing clones argues response in such heterochronous combinations. against the assumption that the testosterone reaction, once initiated, spreads within the mesenchyme independently of Discussion the hormone. "Recognition" of Mammary Epithelium. In male fetuses The recombination experiments with trypsin-isolated, the androgen-induced mesenchymal condensations form mesenchyme-free epithelium and mesenchyme of normal only around the mammary buds, and only mammary epithe embryos and of androgen-insensitive embryos suggest that lium becomes necrotic. The adjacent epidermis with the testosterone acts directly on the mesenchyme. Although an first hair rudiments remains apparently unaffected. additional direct action on the epithelium cannot strictly be We tested the capability of 12-day mammary (dermal?) excluded, this would seem unlikely in view of the typical mesenchyme to discriminate between mammary and var androgen response observed in combinations involving ious other types of epithelium by combining it with mesen- androgen-insensitive epithelium. Necrosis of the mammary chyme-free epithelia of the 11-day pancreas and lung and epithelium as seen in 14-day male fetuses therefore does of the 13-day salivary gland. When testosterone was applied not represent hormone-induced cell death, but rather it to these cultures, no mesenchymal reaction was observed represents hormone-induced cell "killing." to occur around these heterogenous epithelia. In a second Although we are still ignorant of the mechanism by which series of experiments, 1 to 5 pieces of 12-day mam testosterone-stimulated mesenchyme causes necrosis of mary epithelium were placed next to such a heterogenous the epithelium, our experiments have provided some in epithelium on top of mammary mesenchyme. Frequently, formation on the initiation of this hormone-induced tissue the 2 different epithelia fused in vitro, but they remained interaction. From the experiments in which a thin layer of distinguishable in histological sections due to different normal mesenchymal cells was left around the mammary staining properties. After the addition of testosterone, a bud, which was then combined with androgen-insensitive mesenchymal reaction (condensation) was found to occur mesenchyme, it appears that all mesenchymal cells re exclusively around mammary epithelium (Fig. 3). No such quired for the testosterone response are already at the reaction was seen around the other epithelium (pancreas, epithelial surface, as early as in 12-day glands. Thus, the lung, or salivary). In the fused epithelia of dual origin, the hormone does not cause a specialized type of mesenchymal mesenchymal cells condensed only around the mammary cell to move to the mammary bud, a possibility that is also portion. ruled out by the observed mesenchymal heterogeneity Responsive Phases. We know from earlier experiments around X""'/X+ heterozygous glands. If a specialized type that the mammary gland is responsive to testosterone only of mesenchymal cell migrated to the mammary bud in during a relatively short period of its development, extend response to testosterone, these glands would be sur ing from late Day 13 to very early Day 15 of gestation (11). In rounded only by androgen-sensitive clones. The absence of view of our finding that testosterone acts on the mesen a testosterone response in combinations in which a thin chyme, which then affects the epithelium, this transient layer of androgen-insensitive mesenchymal cells separated responsiveness of the entire rudiment need not necessarily the epithelium from a large mass of normal mesenchyme reflect an equally short phase of hormone responsiveness also reinforces this conclusion. The result of this experi of the mesenchyme. It is also conceivable that hormone- ment also indicates that the mesenchymal reaction can only activated mesenchyme "attacks" mammary epithelium only be initiated at the epitheliomesenchymal interface and sug in a certain developmental stage. gests that the mesenchymal cells starting the reaction require 2 "signals." testosterone and close vicinity to (or We had assumed that experimental combinations of epi thelium and mesenchyme derived from embryos differing in contact with?) mammary epithelium. As we have shown, their developmental stage ("heterochronic" combinations) the mesenchyme is able to distinguish between mammary

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Autonomous Variants of Shionogi Mammary Carcinoma Based on Het- epithelium and at least 4 other types of epithelium: epider erogenous Patterns of Androgen Binding. Cell, 73.-273-280, 1978. mis, salivary gland, lung, and pancreas. 3. Drews, U., and Alonso-Lozano, V. X-lnactivation Pattern in the Epididy- We only very rarely observed an androgen response when mis of Sex-Reversed Mice Heterozygous for Testicular Feminization. J. recombination of mammary epithelium with underlying Embryol. Exptl. Morphol., 32: 217-225, 1974. 4. Engelsman. E., Korsten, C. B., Persign, J. P., and Clenton, F. J. mesenchyme was done on Days 13 or 14 of embryonic Estrogens and Androgen Receptors in Human Breast Cancer. Brit. J. development (i.e., just before or during the responsive Cancer, 30. 177, 1974. 5. Grobstein, C. Inductive Epithelio-mesenchymal Interaction in Cultured phase, respectively). The same experiment done on Day 12 Organ Rudiments of the Mouse. Science, 778: 52-55, 1953. consistently gave positive results. In such recombinations, 6. Horwitz, K. B., Costlow, M. E., and McGuire, W. L. MCF-7; A Human it is technically impossible to place the mammary epithelia Breast Cancer Cell Line with Estrogen. Androgen, Progesterone, and Glucocorticoid Receptors. Steroids, 26: 785-796, 1975. exactly on those few mesenchymal cells that had previously 7. Jensen, E. V., and DeSombre, E. R. Estrogens and Progestins. In: G. (i.e., before tissue separation) been in contact with a gland Litwack (ed.), Biochemical Actions of Hormones, Vol. 2, pp. 215-255. bud. In these experiments a new association of mammary New York: Academic Press, Inc., 1972. 8. Kratochwil, K. Organ Specificity in Mesenchymal Induction Demon epithelium with mesechyme of the mammary region is strated in the Embryonic Development of the Mammary Gland of the created. Conceivably, either this association has to persist Mouse. Develop. Biol.,20: 46-71, 1969. 9. Kratochwil, K. In Vitro Analysis of the Hormonal Basis for the Sexual for at least 48 hr before hormone exposure, or the recogni Dimorphism in the Embryonic Development of the Mouse Mammary tion of mammary epithelium by dermal mesenchyme occurs Gland. J. Embryol. Exptl. Morphol., 25: 141-153. 1971. before Day 13. In any event the result suggests that the 10. Kratochwil, K. Experimental Analysis of the Prenatal Development of the Mammary Gland. Mod. Probi. Pediat., 75. 1-15, 1975. mesenchymal cells starting the androgen reaction need to 11. Kratochwil, K. Development and Loss of Androgen Responsiveness in be in close contact with mammary epithelium some time the Embryonic Rudiment of the Mouse Mammary Gland. Develop. Biol., before they become responsive to the hormone. 6Ã•:358-365, 1977. 12. Kratochwil, K., and Schwartz, P. Tissue Interaction in Androgen Re Our current and rather limited understanding of the sponse of Embryonic Mammary Rudiment of Mouse: Identification of androgen response of the embryonic mammary rudiment of Target Tissue for Testosterone. Proc. Nati. Acad. Sei. U. S., 73: 4041- 4044, 1976. the mouse is that this process involves multiple and recip 13. Lippman. M., BolÃ¡n. G.. and Huff, K. The Effects of Androgens and rocal epitheliomesenchymal tissue interaction. First, the Antiandrogens on Hormone-Responsive Human Breast Cancer in Long- epithelial bud exerts some influence on adjacent mesenchy Term Tissue Culture. Cancer Res.. 36: 4610-4618, 1976. 14. Lippman, M. E.. and Huff. K. A Demonstration of Androgen and Estrogen mal cells, which seems to be a prerequisite for their (later) Receptors in Human Breast Cancer Using a New Protamine Sulfate response to testosterone. Once exposed to the hormone, Assay. Cancer, 38: 868-874. 1976. 15. Lyon, M. F. Gene Action in the X-chromosome of the Mouse (Mus these mesenchymal cells condense around the epithelial musculus L.). Nature, 790: 372-373, 1961. bud and, through a yet unknown mechanism, cause the 16. Lyon. M. F.. and Hawkes, S. G. X-linked Gene for Testicular Feminization eventual destruction of mammary epithelium. in the Mouse. Nature, 227: 1217-1219, 1970. 17. Maass, H., Trams, G., Nowakowski, H., and Stolzenbach, G. Steroid Whether these findings have any relevance for the ob Hormone Receptors in Human Breast Cancer and the Clinical Signifi served androgen response of mammary carcinomas is at cance. J. Steroid Biochem.. 6: 743-749, 1975. present unclear. The presence of androgen receptors in 18. Ohno, S., Geller, L. N., and Young Lai, E. V. Tfm Mutation and Masculinization versus Feminization of the Mouse Central Nervous epithelial cell lines (2, 6, 13) certainly points to the epithe System. Cell, 3: 235-242, 1974. lium as the target tissue for the hormone in these cases. 19. Quadri, S. K., Kledzik, G. S., and Meites, J. Enhanced Regression of DMBA-induced Mammary Cancers in Rats by Combination of Ergocor- Our experiments, however, have shown not only that testos ninewith Ovariectomy or High Doses of Estrogen. Cancer Res., 34: 499- terone can act on the mesenchyme of the gland, but also 501,1974. that its effect on the epithelium is also mediated by this 20. Raynaud, A. Effet des Injections d'Hormones Sexuelles Ã la Souris mesenchyme. Obviously, any kind of a comparable andro- Gravide sur le DÃ©veloppementdes Ã‰bauchesdela Glande Mammaire des Embryons. I. Action des Substances AndrogÃ¨nes. Ann. Endocrinol gen-induced tissue interaction in the adult gland or in Paris, S: 248-253, 1947. mammary carcinomas would be of fundamental importance 21. Raynaud, A. Foetal Development of the Mammary Gland and Hormonal Effects on Its Morphogenesis. In: I. R. Falconer, (ed.), Lactation. for an endocrine treatment of metastatic breast cancer. Just Proceedings, International Symposium, Nottingham, pp. 3-29. London: as in our combination experiments, a distant metastasis of Butterworth & Co., 1971. 22. Raynaud, A., and Raynaud. J. Les Principales Ã‰tapesdela SÃ©paration, a mammary carcinoma represents a secondary, nonphysio- d'avec l'Ã‰piderme,des Ã‰bauchesMammaires des Foetus MÃ¢lesde logical association of mammary epithelium with nonmam- Souris: Recherches sur les Processus de la Rupture de la Tige du mary mesenchyme. Such a situation would undoubtedly Bourgeon Mammaire. Compt. Rend. Soc. Biol. 147: 1872-1876, 1953. 23. Smith, J. A., and King, R. J. Effects of Steroids on Growth of an alter the nature of the epitheliomesenchymal interaction Androgen-Dependent Mouse Mammary Carcinoma in Cell Culture. and thereby affect the response to the hormone. Exptl. Cell Res., 73: 351-359, 1972. 24. Teller, M. N., Stock, C. C., Stohr. G., Merker. P.C.. Kaufman, R. J , References Escher, G. C., and Bowie, M. Biologic Characteristics and Chemother apy of 7,12-Dimethylbenz(a)anthracene-induced Tumors in Rats. Cancer 1. AMA Committee on Research. Androgens and Estrogens in the Treat Res., 26: 245-254, 1966. ment of Disseminated Mammary Carcinoma. J. Am. Med. Assoc., 172: 25. Turner, C. W., and Gomez, E. T. The Normal Development of the 1271-1274, 1960. Mammary Gland of the Male and Female Albino Mouse. I. Intrauterine 2. Bruchovsky, N., and Rennie, P.S. Classification of Dependent and Bull. Missouri Agr. Exptl. Sta., 782: 3-20, 1933.

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Fig. 1. Mammary gland rudiment of a 14-day male mouse embryo exhibiting the characteristic androgen response. Mesenchymal cells are seen to condense around the epithelial bud. which later separates from the epidermis and becomes necrotic. 1 /urnEpon sections stained withtoluidine blue, x 400. Fig. 2. Mammary rudiment of an X'""/X' heterozygous female embryo exposed to testosterone for 35 hr in culture. A condensation of mesenchymal cells is seen only on the left side of the epithelium, whereas the remaining mesenchyme appears unaffected. It is assumed that the condensing cells represent an X' phenoclone (i.e., being androgen-sensitive), whereas mesenchymal cells on the right side are androgen-insensitive due to expression of the X'"" chromosome. 1 /j.m Epon sections stained with toluidine blue, x 650. Fig. 3. Mammary mesenchyme simultaneously combined with mammary epithelium (center) and with pancreas epithelium. After 30 hr of testosterone exposure, the characteristic mesenchymal condensation is formed exclusively around mammary epithelium. Pancreas epithelium, even in close vicinity to this condensation, does not provoke such a response. 1 nm Epon sections stained with toluidine blue, x 600.

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Heidelinde Dürnberger, Barbara Heuberger, Peter Schwartz, et al.

Cancer Res 1978;38:4066-4070.

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