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Endocrinology Vol. 139, No. 2 753-764
Copyright © 1998 by The Endocrine Society

ARTICLES

Almost Exclusive Androgenic Action of Dehydroepiandrosterone in the Rat Mammary Gland

Antigone Sourla, Céline Martel, Claude Labrie and Fernand Labrie

Medical Research Council (MCR) Group in Molecular Endocrinology, CHUL (Centre Hospitalier de l’ Université Laval) Research Center and Laval University, Québec, G1V 4G2, Canada

Address all correspondence and requests for reprints to: Professor Fernand Labrie, Medical Research Council (MRC) Group in Molecular Endocrinology, CHUL (Centre Hospitalier de l’ Université Laval) Research Center, 2705 Laurier Boulevard, Québec, Canada G1V 4G2.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine the relative role of the androgenic and/or estrogenic components of the action of dehydroepiandrosterone (DHEA) on the histomorphology and structure of the rat mammary gland, ovariectomized (OVX) female animals received DHEA administered alone or in combination with the pure antiandrogen flutamide or the pure antiestrogen EM-800 for 12 months. We have also evaluated the effect of estradiol (E2) and dihydrotestosterone constantly released from SILASTIC brand silicon implants as well as medroxyprogesterone acetate released from poly(lactide-co-glycolide) microspheres. While 1-yr OVX resulted in a severe atrophy of the mammary gland, treatment of OVX animals with DHEA stimulated lobuloalveolar and ductal growth, as well as the secretory activity of the acinar cells, thus resulting in a lobuloalveolar type of development of the mammary gland. The addition of FLU to DHEA almost completely prevented the stimulatory effect observed with DHEA alone, whereas addition of the antiestrogen EM-800 had no significant effect on the action of DHEA on the mammary gland. At the doses used, medroxyprogesterone acetate and dihydrotestosterone also stimulated ductal and alveolar development, although to a lesser degree than that achieved with DHEA. The stimulatory effect of estradiol was mainly expressed on ductal growth with a smaller stimulatory effect on lobuloalveolar development. The above-indicated stimulatory effects on lobuloalveolar development were also reflected in significant increases of the total and parenchymal gland surface areas of the mammary gland. The present study shows that androgens induce a marked lobuloalveolar type of development of the mammary gland in the rat. Moreover, these data indicate the highly predominant or almost exclusive androgenic component in the potent stimulatory action of DHEA on the histomorphology and structure of the rat mammary gland. In fact, blockade of the potential estrogenic component of DHEA action by EM-800 did not affect the stimulatory action of DHEA on mammary gland histomorphology, whereas the antiandrogen FLU almost completely blocked the effect of DHEA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEX STEROID hormones play an essential role in the morphogenesis, development, growth, and function of the mammary gland in both man (1, 2, 3, 4, 5, 6) and laboratory rodents such as rats and mice (7, 8, 9, 10, 11, 12, 13, 14). Thus, during embryonic development, androgens and especially testosterone cause the involution of the mammary gland of male mouse fetuses (8), whereas a premature development of the mammary gland takes place under the influence of estrogens in animals of both sexes when injected in the pregnant mouse or directly into the embryo (13, 15).

Although mammary gland histology and structure do not differ significantly in young male and female rats (16), the first estrous cycle in female Sprague-Dawley rats results in a rapid growth and differentiation of the mammary gland, a change that can be prevented by ovariectomy (17). In fact, the rat mammary gland is a highly hormone-sensitive tissue (18, 19). In addition, it has been demonstrated that not only ovarian hormones but also mammotrophic hormones of anterior pituitary and of adrenal origin as well as local factors play an important role in the modulation of proliferation and differentiation of the mammary tissue in vivo and in vitro (14, 20, 21, 22).

The rat mammary gland has been widely used as model of hormone-sensitive breast cancer in women (23, 24, 25). On the other hand, androgens have been successfully used for the treatment of breast cancer in women, achieving an objective response comparable to other hormonal therapies (26, 27, 28, 29). In addition, it has been shown that androgens such as dromostanolone propionate and testosterone and dehydroepiandrosterone (DHEA), a precursor of androgens (30, 31), exert a potent inhibitory effect on the development of DMBA-induced mammary carcinoma in the rat (23, 32, 33, 34).

Although DHEA and its sulfate DHEA-S of adrenal origin represent a major source of active sex steroids through their intracrine conversion into potent androgens and estrogens in peripheral tissues (31, 35), their physiological role remains largely unknown. On the other hand, despite the fact that a series of studies have shown the chemopreventive effect of DHEA on the development of rat mammary cancer (23, 32, 36), little is known about the effect of long-term administration of DHEA on mammary gland physiology and structure.

We have used the ovariectomized (OVX) female Sprague-Dawley rat model to investigate the potential effect of DHEA and its active metabolites on the mammary gland histomorphology and structure in adult virgin female rats. We have also compared the effect of DHEA with that of estradiol, medroxyprogesterone, as well as the nonaromatizable androgen dihydrotestosterone (DHT), and we have also used the pure antiandrogen flutamide (FLU) and the pure antiestrogen EM-800 to assess the specific androgenic and/or estrogenic actions of DHEA in the rat mammary gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult female Sprague-Dawley rats [Crl:CD(SD)Br] (Charles River Laboratory, St.-Constant, Canada), aged approximately 2 months and weighing 180–200 g at start of treatment, were used. The rats were acclimated to the environmental conditions (temperature at 22 ± 2 C, 14-h light, 10-h dark cycles, lights on at 0715 h) for at least 1 week before starting the experiment. The animals were housed two per cage and were allowed free access to tap water and a commercial pellet diet (Agway ProLab R-M-H 4018). The experiment was conducted in a Canadian Council on Animal Care-approved facility in accordance with the CCAC Guide for Care and Use of Experimental Animals.

Sixty-four rats were randomly distributed into eight groups of eight animals each as follows: 1) intact control; 2) OVX control; 3) OVX + medroxyprogesterone acetate (MPA); 4) OVX + 17ß-estradiol (E2); 5) OVX + DHT; 6) OVX + DHEA; 7) OVX + DHEA + FLU; 8) OVX + DHEA + EM-800. On the first day of the experiment, the animals of the appropriate groups underwent bilateral OVX under isoflurane-induced anesthesia and one SILASTIC brand silicon implant (Dow Corning, Midland, MI) of E2 or DHT was inserted sc in the dorsal area of each animal of the indicated groups. Implants had the following sizes and concentrations: E2: [cholesterol (1:250, wt:wt), 0.5 cm (length), 0.125 inch (outer diameter), and 0.062 inch (inner diameter)]; DHT: [cholesterol (30:100, wt:wt), 2.5 cm (length), 0.125 inch (outer diameter) and 0.062 inch (inner diameter)]. During the course of the experiment, the implants were replaced every 4–6 weeks. MPA was released from poly(lactide-co-glycolide) microspheres (30 mg) injected sc every 3 months in 2% carboxymethylcellulose, 1% Tween-80 and water. Treatment with the antiandrogen FLU (4'-nitro-3'-trifluoremethylisobutyranilide) (7.5 mg, injected sc twice daily), the antiestrogen EM-800 ((+)-7-pivaloyloxy-3-(4'-pivaloyloxyphenyl)-4-methyl-2-(4''-(2`''-piperidinoethoxy)phenyl)-2H-benzopyran) (250 µg, per os, once daily) (37, 38, 39), and DHEA (30 mg, percutaneous application, twice daily on an approximately 3 cm x 3 cm shaved area of dorsal skin) was initiated on the morning of day 1 of the experiment (40). FLU and EM-800 were administered in 4% ethanol, 4% polyethylene glycol-600, 1% gelatin and 0.9% NaCl, and DHEA was administered in 50% ethanol-50% propylene glycol.

Histology
After 12 months of treatment, the animals were killed by exsanguination from the abdominal aorta under isoflurane anesthesia. The mammary glands were then removed and immediately immersed in a solution of 10% buffered formalin for 48 h. After fixation, mammary gland tissue was processed in a tissue processor and embedded in paraffin blocks. Sections of 5-µm thickness were prepared and stained with hematoxylin-eosin. Histopathologic examination of tissue slides was performed by light microscopy.

Whole-mount preparation
Mammary glands were carefully excised, dissected free from the epidermal layer, stretched onto slides, and immersed in 25% glacial acetic acid in EtOH for 16 h. After fixation, slides were washed in 70% EtOH and distilled water and stained with Carmine Alum overnight. Slides were then dehydrated in increasing concentrations of EtOH (70–100%) in xylene. Examination was then performed under a stereoscope and a light microscope after mounting with Permount glue (Fisher Scientific, Ltd., Nepean, Ontario, Canada) (41).

Quantitative analysis
The total as well as the parenchymal surface areas of the abdominal mammary gland of each animal were measured by tracing the gland with a stylus in the whole mount preparation and projection on a digitizer tablet of a Bioquant Morphometry System (Bioquant Meg IV System, RLM, Biometrics Corp., Nashville, TN) and a SummaSketch (Summagraphics, now owned by Calcomp Technology, Inc., Anaheim, CA) digitalizing tablet in conjunction with a Leitz Aristoplan (Leica Microsystems Canada, Inc., Montreal, Québec, Canada) microscope as previously described (42). In addition, the number of ducts and lobuloalveolar structures present per/mm2 of total surface area of the mammary gland was measured using the same Bioquant morphometry system. The 5-µm sections obtained at different levels of the mammary gland were analyzed from each of the eight animals per group.

Statistical analysis
Data are expressed as the means ± SEM of data obtained from eight animals per group. Statistical significance was determined according to the multiple-range test of Duncan-Kramer (43).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histology and whole mount preparation
The mammary gland of intact female rats aged approximately 14 months at the end of the experiment shows a mild to moderate lobular hyperplasia compared with young adults. The histological pattern is characterized by a large number and increased size of the lobular structures (Fig. 1AGo). In this and all other figures, the results shown are representative of the pattern seen in all animals of each group. The alveoli consist of foamy acinar cells mainly filled with clear secretory vacuoles (Fig. 2AGo). In addition, cystic dilatation of both the mammary ducts and alveolar lumen by eosinophilic secretory material is observed (Fig. 2AGo). In whole mount preparations, the tubuloalveolar pattern of development of the mammary gland is characterized by a marked ductal branching with many alveolar buds (ABs), the latter being organized in well developed, hypertrophic lobules (Fig. 3AGo).



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  		Figure 1. Mammary gland histology in (A) intact control, (B) OVX control, and OVX rats treated with (C) MPA microspheres, (D) estradiol implants, (E) DHT implants, (F) DHEA, 30 mg, cutaneous application, twice daily, on an area of 3 x 3 cm of dorsal skin, (G) DHEA, 30 mg, cutaneous application, twice daily + FLU, 7.5 mg, sc, twice daily and (H) DHEA, 30 mg, cutaneous application, twice daily + EM-800, 250 µg, orally, once daily. An increase in the number of alveolar units (a) was observed in OVX animals treated with MPA (C) and DHT (E) with the formation of small primitive lobules (l) after DHT administration (E). Estradiol treatment induced an increased number of ducts (d), accompanied by the presence of alveolar units (a) and small lobules (l), without evidence of secretory activity (D). A marked increase in the amount of lobuloalveolar tissue (l) and in the secretory activity of the acinar cells accompanied by accumulation of secretory material (s) in the duct lumen (d) were observed after DHEA administration (F). The stimulatory effect of DHEA on the mammary gland was completely blocked by simultaneous treatment with FLU (G), whereas no significant histological change was seen after the addition of EM-800 to DHEA compared with DHEA alone (H). Compare with intact (A) and OVX (B) controls. Hematoxylin-eosin, magnification x200(d, ducts; a, alveoli, l, lobules).

 


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  		Figure 2. Greater magnification showing the histological characteristics of acinar epithelial cells in (A) intact control, (B) OVX control, and OVX rats treated with (C) DHEA, 30 mg, cutaneous application, twice daily, on an area of 3 x 3 cm of dorsal skin, (D) DHEA, 30 mg, cutaneous application, twice daily + FLU, 7.5 mg, sc, twice daily and (E) DHEA, 30 mg, cutaneous application, twice daily + EM-800, 250 µg, orally, once daily. Note the hypertrophy as well as the marked accumulation of mainly clear secretory vacuoles (cv) in the cytoplasm of acinar cells of the mammary gland in DHEA-treated OVX animals (C). After FLU administration (D), both acinar cells of the few remaining regressed alveoli (arrowhead) and epithelial cells lining the ducts (d), showed a foamy cytoplasm filled with only a yellowish-brownish material (arrows). On the other hand, after the addition of EM-800 to DHEA treatment (E), the acinar cells were hypertrophic and filled with a significant amount of mainly eosinophilic secretory vacuoles (ev), a pattern similar to that seen in animals treated with DHEA alone (C). Numerous clear secretory vacuoles (sv) in the cytoplasm of acinar cells and the presence of secretion in the dilated ductal lumen (d) were observed in intact controls (A), whereas in OVX control animals, the ducts (d) are lined by atrophic, inactive, and low cuboidal epithelial cells. Hematoxylin-eosin, magnification x400. The data shown in this and the following figures are representative of the effects observed in all animals of each group.

 


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  		Figure 3. Whole mount preparation of the mammary gland in (A) intact control, (B) OVX control, and OVX rats treated with (C) MPA microspheres, (D) estradiol implants, (E) DHT implants, (F) DHEA, 30 mg, cutaneous application, twice daily, on an area of 3 x 3 cm of dorsal skin, (G) DHEA, 30 mg, cutaneous application, twice daily + FLU, 7.5 mg, sc, twice daily and (H) DHEA, 30 mg, cutaneous application, twice daily + EM-800, 250 µg, orally, once daily. A slight increase in ductal branching (D), with the occasional presence of small ABs, was observed after MPA administration (C), whereas estradiol treatment (D) induced a more pronounced increase in lateral branching (arrows) accompanied by the formation of small ABs. Treatment with DHT (E) promoted the growth of ducts (D) and ABs, the latter being organized into lobules (L). DHEA administration completely reversed the atrophic changes of the mammary gland seen 12 months after ovariectomy and induced a marked stimulation of lobuloalveolar growth (L) of the mammary gland (F). FLU addition to DHEA treatment completely blocked the stimulatory effect of DHEA on the mammary gland (G), whereas no significant histological changes of the structure of the mammary gland were observed after simultaneous administration of DHEA and EM-800 compared with DHEA alone (H). Carmine Alum, magnification x60.

 
Twelve months after ovariectomy, a severely atrophic mammary gland is observed (Fig. 1BGo); it contains only small atrophic ducts lined by a flattened atrophic epithelium (Fig. 2BGo). No acinar or lobular structure is seen. In the whole mount preparation, the mammary gland consists only of a few primary, secondary, and tertiary ducts with a few lateral buds, whereas no ABs or lobular structures can be seen (Figs. 3BGo and 4AGo).



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  		Figure 4. Whole mount preparation of the mammary gland in (A) OVX control and OVX rats treated with (B) MPA microspheres, (C) estradiol implants, (D) DHT implants, (E) DHEA, 30 mg, cutaneous application, twice daily, on an area of 3 x 3 cm dorsal skin, (F) DHEA, 30 mg, cutaneous application, twice daily + FLU, 7.5 mg, sc, twice daily and (G) DHEA, 30 mg, cutaneous application, twice daily + EM-800, 250 µg, orally, once daily. Greater magnification showing the slight increase in the number of lateral buds (large arrowheads) and terminal end buds (small arrowheads) as well as ABs after MPA administration (B); estradiol treatment (C) induced a more pronounced increase in lateral branching (D), whereas the presence of lobuloalveolar units (L) and more ABs were seen after DHT administration (D). DHEA treatment (E) induced an increase in ductal growth and in both the number and size of lobules (L), an effect that was abolished by the addition of FLU (F) where mainly terminal ducts and terminal end buds were seen (small arrowheads). After treatment of OVX animals with both DHEA and EM-800 (G), the structure of the mammary gland did not differ from that seen after treatment with DHEA alone, the pattern being characterized by the predominant presence of lobuloalveolar units (L). Carmine Alum, magnification x100

 
Treatment of OVX animals with MPA did not result in major histological changes of the mammary gland, which remained moderately atrophied. We could observe, however, a slight increase in the number of alveolar units that are composed of small alveoli lined by a single layer of low cuboidal epithelial cells containing clear and/or eosinophilic cytoplasmic vacuoles (Fig. 1CGo). In the whole mount preparation, a more developed duct system compared with OVX controls is observed. These MPA-induced changes are characterized by an increased number of secondary and tertiary ducts with the predominance of lateral buds, terminal end buds (TEBs), and terminal ducts (TDs). Occasionally, small ABs can also be seen (Figs. 3CGo and 4BGo).

A partial reversal of the marked atrophy of the mammary gland observed 12 months after ovariectomy was seen after estradiol treatment of OVX animals (Fig. 1DGo). The estrogenic effect is characterized by the induction of a tubuloalveolar type of development. A well developed duct system with clusters of alveoli forming a few small lobular structures is seen. The ductal as well as the alveolar epithelium consist of low, cuboidal epithelial cells, without evidence of increased secretory activity (Fig. 2DGo). In the whole mount preparation, estradiol administration is seen to induce mainly the development of the duct system (Fig. 3DGo). Thus, a significant increase in ductal length as well as ductal thickness and lateral branching is observed, compared with OVX controls, as well as to animals treated with MPA. An increase in the number of tertiary ducts is also seen, with many lateral and terminal end buds, the latter giving rise to ABs that are organized into small primitive lobules (Figs. 3DGo and 4CGo).

DHT treatment, on the other hand, induces a marked increase in the number of lateral buds, TEBs, TDs, and ABs (Fig. 1EGo). As well illustrated in the whole mount preparation, this effect of DHT is more pronounced than that observed after estradiol administration (Fig. 1DGo). In addition, the presence of small lobular structures is also observed (Figs. 3EGo and 4DGo). Histologically, the mammary gland is composed of an increased number of small lobuloalveolar units and the ovariectomy-induced atrophy is thus partially reversed at the dose used. In addition, the alveoli are lined by hypertrophic eosinophilic acinar cells containing secretory vacuoles (Fig. 1EGo).

As illustrated in Fig. 1FGo, a complete reversal of the mammary gland atrophy caused by castration is seen after DHEA administration to OVX animals. A profuse lobular growth is observed, the mammary gland being composed of a well developed duct system and a large number of lobular structures exhibiting a typical lobuloalveolar type of development. In addition, a mild to moderate lobular hyperplasia is observed after DHEA treatment with an increased number and size of lobular structures. These numerous lobular structures are lined by hypertrophic acinar cells filled with mainly eosinophilic and clear secretory vacuoles, displacing laterally or basally the small darkly stained nuclei. Occasionally, the alveolar lumen is filled with secretory material and a mild dilatation of ducts can be seen (Fig. 2CGo). In whole mount preparations, an increase in lateral branching, and mainly in the number and size of the ABs and lobules is observed, with a consequent significant increase in the amount of lobuloalveolar tissue (Figs. 3EGo and 4DGo).

The addition of FLU to DHEA treatment resulted in an almost complete prevention of the DHEA-induced histological changes of the mammary gland (Fig. 1GGo). The mammary gland was then mainly composed of the duct system, with only occasional remaining small lobules consisting of alveoli lined by low cuboidal epithelial cells. The presence of brownish-yellowish material is also seen in the cytoplasm of both acinar cells and cells lining the ducts, thus giving a foamy appearance to the epithelial cells (Fig. 2DGo). In whole mount preparations, the mammary gland consist of primary, secondary, and tertiary ducts and TEBs with a marked decrease in lateral branching as well as in the number of ABs compared with DHEA alone. No formation of lobular structures could be seen (Figs. 3GGo and 4FGo). It is also of interest to note that although FLU prevented the changes induced by DHEA treatment, the mammary gland did not reach the severe atrophy seen in control OVX animals 12 months after castration.

On the other hand, following combined treatment with DHEA and EM-800, no significant histological changes were observed compared with those seen in OVX animals treated with DHEA alone. The mammary gland was composed of a well developed duct system, with a large number of well developed lobular structures presenting a lobuloalveolar type of development (Fig. 1HGo). In addition, a mild to moderate lobular hyperplasia was observed, this pattern being characterized by an increased number and size of lobular structures, as seen in OVX animals treated with DHEA alone. A marked hypertrophy and eosinophilia of the epithelial cells lining the alveoli is also noted, this being accompanied by a significant accumulation of mainly eosinophilic and clear secretory vacuoles in the cytoplasm (Fig. 2EGo). In the whole mount preparations, the structure of the mammary gland is characteristic of a lobuloalveolar type of development, analogous to that seen in animals treated with DHEA alone; a significant increase in lateral branching, with the predominant presence of hypertrophic lobuloalveolar units are seen (Fig. 3H, 4G).

Quantitative analysis
Ovariectomy resulted in a dramatic decrease in the total as well as parenchymal surface areas of the mammary gland s compared with intact controls (Figs. 5Go and 6Go). After treatment with estradiol, significant increases of the total and parenchymal surface areas of the mammary gland were observed from 55 ± 3.5 mm2 to 450 ± 48.5 mm2 (P < 0.01) and from 5.5 ± 2.1 to 196 ± 46.1 mm2 (P < 0.01), respectively. After MPA administration, increases of the total and parenchymal surface areas of the mammary gland to 75 ± 10.4 mm2 and 28.5 ± 13 mm2 were observed, respectively (P < 0.05). On the other hand, at the dose used, DHT treatment resulted in a more important stimulation of the above-indicated parameters that increased to 550 ± 76.5 mm2 (P < 0.01) and to 255 ± 32.5 mm2 (P < 0.01), respectively (Figs. 5Go and 6Go). In the same figures, it can be seen that DHEA treatment induced a marked increase in both the total and parenchymal surface areas to 1680 ± 214 mm2 (P < 0.01) and 1200 ± 125 mm2 (P < 0.01), respectively.



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  		Figure 5. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA + FLU or EM-800 on total mammary gland surface area (*, P < 0.05; **, P < 0.01 vs. OVX; ##, P < 0.01, vs. DHEA).

 


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  		Figure 6. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA + FLU or EM-800 on parenchymal surface area of the mammary gland (*, P < 0.05; **, P < 0.01 vs. OVX; ##, P < 0.01, vs. DHEA).

 
Interestingly, after DHEA administration, the parenchymal to stromal ratio of the mammary gland was also significantly greater than that observed in intact animals (Fig. 7Go). All the above-described stimulatory effects of DHEA were almost completely reversed by concomitant treatment with FLU, which returned the total and parenchymal surface areas to values not significantly different from those measured in OVX control animals. On the contrary, the administration of the pure antiestrogen EM-800 in combination with DHEA had no influence on the effect of DHEA on the parameters measured ( Figs. 5–7GoGoGo).



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  		Figure 7. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA + FLU or EM-800 on the parenchymal/stromal surface area ratio.

 
Ovariectomy, on the other hand, resulted in a complete absence of lobuloalveolar structures from 1.9 ± 0.2 to 0.0 ± 0.0 per mm2 of total surface area of the mammary gland compared with intact controls (P < 0.01) (Fig. 8Go), whereas the number of ducts was markedly decreased from 2.4 ± 0.2/mm2 to 1.0 ± 0.0 mm2 (P < 0.01) after ovariectomy.



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  		Figure 8. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA + FLU or EM-800 on the number of lobuloalveolar units per mm2 of total surface area of the mammary gland (*, P < 0.05; **, P < 0.01 vs. OVX; ##, P < 0.01, vs. DHEA).

 
Treatment with DHEA induced significant increases in the number of lobuloalveolar structures and ducts to 0.8 ± 0.2/mm2 and 4.2 ± 0.4/mm2, respectively (Figs. 8Go and 9Go). The effect of DHEA on the number of lobuloalveolar units was completely abolished by the concomitant administration of FLU, whereas the addition of EM-800 had no effect to the action of DHEA (Fig. 8Go). On the other hand, administration of E2 resulted in a significant increase of the number of ducts per mm2 of total surface area to 2.6 ± 0.2/mm2 from 1.0 ± 0.0/mm2 in OVX controls (P < 0.01) (Fig. 9Go). Treatment with MPA and DHT also stimulated ductal growth from 1.0 ± 0.0/mm2 to 1.6 ± 0.2/mm2 (P < 0.05) and 1.8 ± 0.2/mm2 P < 0.05), respectively, although the effect was inferior to that achieved with E2 (Fig. 9Go).



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  		Figure 9. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA + FLU or EM-800 on the number of ducts per mm2 of total surface area of the mammary gland (*, P < 0.05; **, P < 0.01 vs. OVX).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study clearly demonstrates the potent stimulatory effect of androgens on mammary gland histomorphology and structure in the female Sprague-Dawley rat. Moreover, the present data show that DHEA treatment not only reversed the atrophic changes of the mammary gland induced by ovariectomy but also enhanced the secretory activity of the epithelial acinar cells and induced a lobuloalveolar type of development of the mammary gland comparable to that seen during pregnancy and lactation (42). The 12-month duration of treatment has been chosen to obtain best assessment of the long-term effects of each treatment.

Mammary gland development, growth, function, and morphology are well known to be dependent upon the endocrine system. Duct and alveolar tissue are structures responsive to hormonal changes observed during the estrous cycle, pregnancy, lactation, and with aging and diet (6, 13, 42). The mammary gland of virgin female rats consists mainly of a well developed duct system including a large number of TEBs, TDs, ABs, and as a few lobules, this pattern being characteristic of a tubuloalveolar type of development. During pregnancy and lactation when the mammary gland reaches its full development and maturity, a profuse lobular development accompanied by a marked stimulation of secretory activity of alveolar epithelium is observed (15, 42).

The multifocal proliferation and the increased secretion of the acinar/alveolar tissue associated with duct dilatation and formation of cysts, seen in the intact animals aged approximately 16 months, represent spontaneously occurring changes during aging in female rats (44, 45). The loss of regular ovarian cyclicity that characterizes aging, especially after 12 months of age, is accompanied by alterations in the serum levels of various hormones, especially estrogens and progestins. More specifically, it has been shown that at 12–19 months of age, female rats enter into a stage of constant estrous accompanied by increased serum estrogen and PRL levels and decreased progesterone concentrations compared with young cycling animals (44, 46, 47). The above-indicated alterations of the hormonal milieu, and especially the increased serum PRL levels, can be correlated with the morphological alterations of the mammary gland seen in aging female rats. It is also well known that the loss of estrous cycles associated with reduced levels of gonadotropins and increased levels of estrogens and PRL are often accompanied by the development of hyperplastic and neoplastic changes of the rat mammary gland (20).

The majority of the intact control animals in our study, aged approximately 15 months, also showed histomorphological signs typical of a chronic anovulatory state, thus explaining the inappropriate secretory activity of the acinar epithelium as well as duct dilatation-ectasia and lobular hyperplasia in the mammary gland. Whereas ovariectomy resulted in a compete atrophy of the mammary gland, estradiol administration mainly induced an increase in duct proliferation and branching with a much less important stimulation of acino-lobular development. The above-indicated stimulatory effects of estradiol on mammary gland development were also reflected in significant increases in the total and parenchymal surface areas of the gland. These results are in agreement with the known effects of estradiol treatment on the mammary gland of female rats (minimal acinar development, maximal stromal, and ductal cell proliferation) (48). In addition, ductal elongation and branching are events known to be under the control of estradiol starting at time of initiation of mammary gland development (48, 49, 50).

At the dose used, MPA administration had minor effects on the duct system. Although at a much lesser degree than that of estradiol, MPA slightly increased the lateral branching as well as the number of TEBs and TDs. Interestingly, a small number of ABs were also observed after MPA administration. The ABs of MPA-treated OVX animals were composed of epithelial cells that contained secretory vacuoles, whereas in OVX controls, the mammary gland only consisted of a few atrophic ducts lined by atrophic, low cuboidal, and inactive epithelium. The above-described histological changes were also accompanied by significant increases in the number of ducts and lobuloalveolar structures present per mm2 of total surface area of the mammary gland as compared with OVX controls.

It is reported that progestins can stimulate the mammary gland of female rats by increasing PRL release (51, 52, 53, 54). Furthermore, high doses of estradiol have been reported to induce cystic mastopathy associated with increased secretory activity, these effects being potentiated by combination with a progestin (55, 56). It is well demonstrated in both rats (57, 58, 59, 60, 61, 62) and mice (63) that the androgenic activity of MPA is exerted through direct interaction with the androgen receptor. Similarly, the histological MPA-induced changes of the mammary gland possibly represent a direct androgen receptor-mediated effect of MPA on the mammary gland.

DHT administration to OVX animals induced an important increase in lateral ductal branching, as well as in the number of TEBs and TDs. In addition, the presence of numerous alveolar structures and lobular units, showing secretory activity, were also seen. At the doses of DHEA and estradiol used, the above-summarized histological changes of the mammary gland induced by DHT treatment were more pronounced than those achieved after estradiol administration. In addition, a significant increase in the total and parenchymal surface areas of the mammary gland as well as in the number of ducts and lobuloalveolar structures present per mm2 of the total surface area of the gland were observed after DHT treatment. Knowing that DHT cannot be aromatized into estrogens, the present data indicate a direct androgenic action.

DHEA is a sex steroid precursor that is metabolized into active androgens and/or estrogens in peripheral intracrine tissues, depending upon the relative activities and types of steroidogenic enzymes expressed in each tissue and cell (31, 35). The mammary gland is likely to possess all the steroidogenic enzymatic systems necessary for the formation of androgens and estrogens from steroid precursors, such as DHEA (64, 65, 66, 67, 68, 69). The complete reversal of the ovariectomy-induced mammary gland atrophy seen after DHEA treatment was characterized by a marked stimulation of the ductal and mainly the lobular structures. In addition, epithelial cell hypertrophy and a marked stimulation of secretory activity were seen, these effects being accompanied by the accumulation of clear and eosinophilic vacuoles in the cytoplasm of the acinar cells. As mentioned above, the above-indicated histological changes characterizing a rather lobuloalveolar type of development of the mammary gland, are analogous to those seen during pregnancy and lactation (14, 70).

In the OVX female Sprague-Dawley rat, exogenous DHEA represents the only source of sex steroids in peripheral tissues, including the mammary gland. It should also pointed out that DHEA does not possess any significant androgenic or estrogenic activity by itself. Thus, the stimulation of lobuloalveolar growth seen after DHEA treatment in OVX animals results from its intracrine in situ conversion into potent androgens and/or estrogens in the mammary gland (31, 67, 68).

It is also noteworthy that, after DHEA treatment, the increase in total gland surface area was mainly due to an increase in the parenchymal surface area, thus resulting in a parenchymal to stromal ratio greater than that observed in intact animals. Furthermore, the observed increase in parenchymal surface area was mainly associated with an increase in the number of lobuloalveolar structures and to a lesser degree by an increase in the number of ducts present per mm2 of total surface area of the mammary gland. Interestingly, the stimulation of lobuloalveolar growth of the mammary gland was almost completely abolished by the concomitant administration of the pure antiandrogen FLU, thus providing evidence for the predominant androgenic effect of DHEA, through its intracrine conversion to active sex steroids with androgenic activity. The mammary gland of OVX animals treated with the combination of DHEA and FLU, although not reaching the severe atrophy seen in OVX control animals, did not demonstrate lobular development. The mammary gland was, in fact, composed of a few ducts and alveolar units without evidence of secretory activity of the epithelial cells. Van Wanegen et al. (71) and Selye et al. (72) have reported the stimulatory effects of androgens, such as testosterone, on lobuloalveolar development in both the rhesus monkey and rat. The lobuloalveolar type of development of the mammary gland is characterized by the predominance of numerous, contiguous lobular structures composed of acinar epithelial cells with abundant, foamy cytoplasm filled with secretory vacuoles, and it is also seen in adult male Sprague-Dawley rats. Moreover, Cardy (44) has reported that the lobuloalveolar structure of the mammary gland seen in male rats can be altered and assume tubuloalveolar characteristics indistinguishable from those seen in adult female rats, after hormonal manipulation with compounds that increase PRL release. In the same report, it was suggested that progestins as well as androgens could stimulate lobuloalveolar growth.

Nevertheless, although it is reported that androgens can stimulate lobuloalveolar growth, our study demonstrates for the first time the stimulatory androgenic-like effect of DHEA on the mammary gland, which not only resulted in a complete reversal of the ovariectomy-induced atrophic changes of the mammary gland but also led to a profuse lobuloalveolar development. In addition, we have also demonstrated the potent stimulatory effect of DHT, a nonaromatizable androgen on the growth of the rat mammary gland, thus indicating that the above-described effects are mediated through the androgen receptor. Furthermore, in the present study, the absence of a significant increase in serum PRL levels in DHEA-treated animals appears to exclude the possibility of a role of PRL in the major DHEA-induced histological changes. Following the combined administration of DHEA and EM-800 to OVX rats, the same lobuloalveolar pattern of development of the mammary gland was seen as that observed after treatment with DHEA alone, thus practically eliminating the role of estrogens in the action of DHEA. It is also important to mention that EM-800 does not have any effect on the mammary gland histopathology when given to OVX rats, as reported for the mouse by Luo et al. (39). The 250-µg daily dose of EM-800 used in the one shown in a series of preclinical pharmacological and toxicological (our unpublished observations) studies (37, 38, 39, 73) to exert maximal antiestrogenic activity.

It is also noteworthy that lobular development and lobular hyperplastic lesions, such as hyperplastic alveolar nodules, often accompanied by enhanced secretory activity (74) are not considered as preneoplastic lesions in the rat (75). The susceptibility and responsiveness of the mammary gland to various exogenous or endogenous hormonal stimuli is modulated by local factors such as the tissue concentration of specific receptors (76). Androgens, on the other hand, are known to be able to alter the concentration of other receptors in mammary tissue, such as progesterone receptors (77). In addition, it has been shown that androgens such as DHT or compounds with androgenic activity, such as MPA, can stimulate 17ß-HSD activity in favor of the formation of estrone from the more potent estrogen estradiol in human breast cancer lines (78). Consequently, alterations of enzymatic activities in the mammary tissue under the influence of locally produced steroids exerting androgenic action, may also account for the observed changes in the structure of the mammary gland.

In conclusion, the present study shows the potent stimulatory effects of androgens on lobuloalveolar as well as ductal development in the rat mammary gland. Furthermore, the histological changes of the mammary gland induced by DHEA treatment provide evidence for its intracrine conversion into active sex steroids with predominant or even possibly exclusive androgenic activity in the mammary gland. Local formation of androgens and estrogens through intracrine activity plays a major role in the pathophysiology of both normal and tumoral hormone-sensitive mammary tissue in the human. Considering the predominant androgenic action of DHEA on normal mammary tissue as well as the well recognized and potent inhibitory action of DHEA on the development and growth of DMBA-induced mammary tumors, which is mainly considered an androgenic effect, we suggest that tissue DHEA metabolism plays an important role in the pathophysiology of the mammary gland and could be a useful preventive and therapeutic approach for breast cancer.

Received June 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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