0013-7227/98/$03.00/0
Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 2
Printed in U.S.A.
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
ABSTRACT
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 EM800 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. (Endocrinology 139: 753–764, 1998)
S
treatment of breast cancer in women, achieving an objective
response comparable to other hormonal therapies (26 –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, 32–34).
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 SpragueDawley 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.
EX STEROID hormones play an essential role in the morphogenesis, development, growth, and function of the
mammary gland in both man (1– 6) and laboratory rodents
such as rats and mice (7–14). 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 –22).
The rat mammary gland has been widely used as model
of hormone-sensitive breast cancer in women (23–25). On the
other hand, androgens have been successfully used for the
Received June 2, 1997.
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.
Materials and Methods
Adult female Sprague-Dawley rats [Crl:CD(SD)Br] (Charles River
Laboratory, St.-Constant, Canada), aged approximately 2 months and
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
weighing 180 –200 g at start of treatment, were used. The rats were
acclimated to the environmental conditions (temperature at 22 6 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 1
medroxyprogesterone acetate (MPA); 4) OVX 1 17b-estradiol (E2); 5)
OVX 1 DHT; 6) OVX 1 DHEA; 7) OVX 1 DHEA 1 FLU; 8) OVX 1
DHEA 1 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(lactideco-glycolide) microspheres (30 mg) injected sc every 3 months in 2%
carboxymethylcellulose, 1% Tween-80 and water. Treatment with the
antiandrogen FLU (49-nitro-39-trifluoremethylisobutyranilide) (7.5 mg,
injected sc twice daily), the antiestrogen EM-800 ((1)-7-pivaloyloxy-3(49-pivaloyloxyphenyl)-4-methyl-2-(40-(290-piperidinoethoxy)phenyl)2
H-benzopyran) (250 mg, per os, once daily) (37–39), and DHEA (30 mg,
percutaneous application, twice daily on an approximately 3 cm 3 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-mm 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-mm sections obtained at different levels of the mammary
gland were analyzed from each of the eight animals per group.
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Statistical analysis
Data are expressed as the means 6 sem of data obtained from eight
animals per group. Statistical significance was determined according to
the multiple-range test of Duncan-Kramer (43).
Results
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. 1A).
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. 2A). In addition, cystic dilatation of
both the mammary ducts and alveolar lumen by eosinophilic
secretory material is observed (Fig. 2A). 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. 3A).
Twelve months after ovariectomy, a severely atrophic
mammary gland is observed (Fig. 1B); it contains only small
atrophic ducts lined by a flattened atrophic epithelium (Fig.
2B). 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.
3B and 4A).
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. 1C). 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. 3C and 4B).
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. 1D). 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. 2D). In the whole mount preparation,
estradiol administration is seen to induce mainly the development of the duct system (Fig. 3D). 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
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
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FIG. 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 3 3 cm of dorsal skin, (G)
DHEA, 30 mg, cutaneous application,
twice daily 1 FLU, 7.5 mg, sc, twice
daily and (H) DHEA, 30 mg, cutaneous
application, twice daily 1 EM-800, 250
mg, 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,magnification3200
(d, ducts; a, alveoli, l, lobules).
terminal end buds, the latter giving rise to ABs that are
organized into small primitive lobules (Figs. 3D and 4C).
DHT treatment, on the other hand, induces a marked
increase in the number of lateral buds, TEBs, TDs, and ABs
(Fig. 1E). As well illustrated in the whole mount preparation,
this effect of DHT is more pronounced than that observed
after estradiol administration (Fig. 1D). In addition, the presence of small lobular structures is also observed (Figs. 3E and
4D). 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. 1E).
As illustrated in Fig. 1F, 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 develop-
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
ment. 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,
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the alveolar lumen is filled with secretory material and a mild
dilatation of ducts can be seen (Fig. 2C). 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. 3E and 4D).
FIG. 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 3 3 cm
of dorsal skin, (D) DHEA, 30 mg, cutaneous application, twice daily 1 FLU,
7.5 mg, sc, twice daily and (E) DHEA, 30
mg, cutaneous application, twice daily
1 EM-800, 250 mg, 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 yellowishbrownish material (arrows). On the
other hand, after the addition of EM800 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 3400. The
data shown in this and the following
figures are representative of the effects
observed in all animals of each group.
FIG. 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 3 3 cm of dorsal skin, (G) DHEA,
30 mg, cutaneous application, twice daily 1 FLU, 7.5 mg, sc, twice daily and (H) DHEA, 30 mg, cutaneous application, twice daily 1 EM-800,
250 mg, 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 360.
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
The addition of FLU to DHEA treatment resulted in an
almost complete prevention of the DHEA-induced histological changes of the mammary gland (Fig. 1G). 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. 2D). 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. 3G and 4F). 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. 1H). 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.
759
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. 2E). 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. 5 and 6). After treatment
with estradiol, significant increases of the total and parenchymal surface areas of the mammary gland were observed
from 55 6 3.5 mm2 to 450 6 48.5 mm2 (P , 0.01) and from
5.5 6 2.1 to 196 6 46.1 mm2 (P , 0.01), respectively. After
MPA administration, increases of the total and parenchymal
surface areas of the mammary gland to 75 6 10.4 mm2 and
28.5 6 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 6 76.5 mm2 (P , 0.01) and to 255 6
32.5 mm2 (P , 0.01), respectively (Figs. 5 and 6). In the same
figures, it can be seen that DHEA treatment induced a
FIG. 5. Effect of ovariectomy and treatment
of OVX rats for 12 months with estradiol,
MPA, DHT, DHEA, and DHEA 1 FLU or
EM-800 on total mammary gland surface
area (*, P , 0.05; **, P , 0.01 vs. OVX; ##,
P , 0.01, vs. DHEA).
FIG. 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 3 3 cm dorsal skin, (F) DHEA, 30 mg, cutaneous
application, twice daily 1 FLU, 7.5 mg, sc, twice daily and (G) DHEA, 30 mg, cutaneous application, twice daily 1 EM-800, 250 mg, 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 3100
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
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FIG. 6. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA 1
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).
FIG. 7. Effect of ovariectomy and treatment
of OVX rats for 12 months with estradiol,
MPA, DHT, DHEA, and DHEA 1 FLU or
EM-800 on the parenchymal/stromal surface area ratio.
marked increase in both the total and parenchymal surface
areas to 1680 6 214 mm2 (P , 0.01) and 1200 6 125 mm2 (P ,
0.01), respectively.
Interestingly, after DHEA administration, the parenchymal to stromal ratio of the mammary gland was also significantly greater than that observed in intact animals (Fig. 7).
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. 5–7).
Ovariectomy, on the other hand, resulted in a complete
absence of lobuloalveolar structures from 1.9 6 0.2 to 0.0 6
0.0 per mm2 of total surface area of the mammary gland
compared with intact controls (P , 0.01) (Fig. 8), whereas the
number of ducts was markedly decreased from 2.4 6 0.2/
mm2 to 1.0 6 0.0 mm2 (P , 0.01) after ovariectomy.
Treatment with DHEA induced significant increases in the
number of lobuloalveolar structures and ducts to 0.8 6 0.2/
mm2 and 4.2 6 0.4/mm2, respectively (Figs. 8 and 9). 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. 8). 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 6 0.2/mm2 from 1.0 6
0.0/mm2 in OVX controls (P , 0.01) (Fig. 9). Treatment with
MPA and DHT also stimulated ductal growth from 1.0 6
0.0/mm2 to 1.6 6 0.2/mm2 (P , 0.05) and 1.8 6 0.2/mm2 P ,
0.05), respectively, although the effect was inferior to that
achieved with E2 (Fig. 9).
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
761
FIG. 8. Effect of ovariectomy and treatment
of OVX rats for 12 months with estradiol,
MPA, DHT, DHEA, and DHEA 1 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).
FIG. 9. Effect of ovariectomy and treatment of OVX rats for 12 months with estradiol, MPA, DHT, DHEA, and DHEA 1
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
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
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
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 –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 (51–54). 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– 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
Endo • 1998
Vol 139 • No 2
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 – 69). The complete reversal of the ovariectomyinduced 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 aboveindicated 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
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ANDROGENIC EFFECT OF DHEA ON THE RAT MAMMARY GLAND
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 SpragueDawley 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-mg daily dose of EM-800 used in the one shown in
a series of preclinical pharmacological and toxicological (our
unpublished observations) studies (37–39, 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 17b-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.
763
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 DMBAinduced 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.
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