==== Front
Exp AnimExp. AnimEXPANIMExperimental Animals1341-13571881-7122Japanese Association for Laboratory Animal Science 2952686617-013410.1538/expanim.17-0134OriginalRelationship between morphological development and sex hormone receptor
expression of mammary glands with age in male rats Miyamoto Yoko 1Kawaguchi Hiroaki 2Tanimoto Akihide 31) Primate Research Institute, Kyoto University, 41-2 Kanrin,
Inuyama-shi, Aichi 484-8506, Japan2) Department of Hygiene and Health Promotion Medicine,
Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka,
Kagoshima-shi, Kagoshima 890-8544, Japan3) Department of Pathology, Kagoshima University Graduate
School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima-shi, Kagoshima
890-8544, JapanAddresses corresponding: H. Kawaguchi, Department of Hygiene and Health Promotion
Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1
Sakuragaoka, Kagoshima-shi, Kagoshima 890-8544, Japan. A. Tanimoto, Department of Pathology,
Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka,
Kagoshima-shi, Kagoshima 890-8544, Japan9 3 2018 2018 67 3 361 371 21 10 2017 20 2 2018 ©2018 Japanese Association for Laboratory Animal
Science2018This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0:

https://creativecommons.org/licenses/by-nc-nd/4.0/)The aim of this study is to investigate the changes with age on morphology and sex
hormone receptor expression in the mammary glands of male Sprague-Dawley rats, focusing on
male-specific cells, “oxyphilic cells”, observed after sexual maturity. The mammary glands
of male rats at 14, 21, 35, 50, 75 and 100 days old were examined by gross observation,
microscopic observation using whole mount specimens, histological and immunohistochemical
sections. Grossly, mammary glands showed brown color at 50–100 days old. In whole mount
specimens, terminal end buds (TEBs) were observed at 14–50 days old and the number of TEBs
was highest at 35 days old. Histologically, the male mammary glands contained small
epithelial cells with scanty cytoplasm at 14–35 days old while ductal and lobular
epithelial cells were changed into oxyphilic cells with abundant cytoplasm at 50–100 days
old. Immunohistochemicaly, androgen receptor (AR), estrogen receptor (ER) and progesterone
receptor (PgR) expressions were found in both mammary glands found at a young age and
oxyphilic cells. In oxyphilic cells, AR expression was dominant compared to ER and PgR
expressions and increased with age. From these results, the development at 50–100 days old
might be strongly related to AR. Ultrastructural observation of oxyphilic cells confirmed
a number of lipid droplets, deformed and/or enlarged mitochondria, lysosomes and
peroxisomes in their cytoplasm.

developmenthormone receptormale ratmammary glandoxyphilic cell
==== Body
Introduction
Breast cancer is the second most common cancer worldwide and the most frequent cancer in
women [7]. Breast cancer in men is rare, with less
than 1% of all breast cancer diagnoses [17], while
the incidence is increasing [10, 34]. Rats have been used for the mammary carcinogenesis
model [13, 15,
27, 31,
36], and several investigators have reported the
development of mammary glands with age in female rats [19, 20, 24, 29]. Little is known about the male
mammary glands in rats except for limited information from a few reports [4, 8, 11, 21, 28]. In particular, male-specific cells in mature male
rats, with abundant and eosinophilic cytoplasm, were described in some reports; however, the
details remain unknown. Additionally, although sex hormone receptors were strongly related
to mammary gland development, only limited information is available on intact male mammary
glands.

More information is necessary to understand the morphological characteristics and hormone
receptor expression in mammary gland development of male rats. The two specific aims of this
study are (1) to investigate morphological changes in the male mammary glands with age from
preweaning to adult stage, especially focusing on male-specific cells called “oxyphilic
cells” [36], and (2) to clarify the relationship
between morphological changes and hormone receptor expression.

Materials and Methods
Ethics statement
The use of animals in this research complied with all relevant guidelines set by
Kagoshima University. The research was performed according to the Institutional Guidelines
for Animal Experiments and in compliance with the Japanese Law Concerning the Protection
and Control of Animal (Law No. 105 and Notification No. 6), and was approved by the Ethics
Committee for Animal Experimentation at Kagoshima University (approval number 2004M00434,
2005M00012, A08054, A09032).

Animals
Inbred Sprague-Dawley (SD) male rats (Rattus norvegicus) used in the
present experiments were bred by self-breeding in Kagoshima University. The animals were
maintained in a filtered air laminar flow room. The room temperature was maintained at 25
± 2°C and the relative humidity at 55% ± 10%, with a 12 h light/dark cycle. The animals
were given a commercial diet (CE-2, CLEA Inc., Tokyo, Japan) and tap water ad
libitum.

All animals were humanely euthanized under deep anesthesia using pentobarbital sodium
(intraperitoneally, Somnopentyl; Kyoritsu Seiyaku Corporation, Tokyo, Japan),
exsanguinated from the abdominal aorta, and necropsied at 14, 21, 35, 50, 75 and 100 days
old (14–35 days old: young age, 50–100 days old: adult age). The number of animals in each
group was five. Mammary glands were grossly observed, and bilateral abdominal mammary
glands were resected and fixed in 10% phosphate-buffered formalin. Additionally, one adult
male rat at 75 days old was used to obtain the samples of mammary glands for transmission
electron microscopy (TEM) examination.

Whole mount specimens
The resected right abdominal mammary glands (gland #4–6) were stained with alum carmine
for 24 h and stored in cedar oil for preparation of whole mount specimens. The specimens
were observed under a stereoscopic microscope, and the terminal end buds (TEBs) in the
distal portions of mammary glands were counted as described previously [9, 27, 35].

Histological and immunohistochemical examination
The resected left abdominal mammary glands were routinely processed for paraffin-embedded
tissue sections. Five µm sections were stained with hematoxylin and eosin
(H&E) for histological evaluation. For immunohistochemistry (IHC), the Dako Envision
Polymer method was used. Paraffin slides were deparaffinized in xylene and rehydrated with
graded ethanol. Antigen retrieval was performed by microwave treatment in Target Retrieval
Solution, pH9.0 (Dako, Tokyo, Japan). Endogenous peroxidase was inactivated by 3% (v/v)
hydrogen peroxidase-methanol solution. The sections were incubated overnight at 4°C with
diluted primary antibodies: mouse monoclonal anti human androgen receptor (AR; Perseus
Proteomics Inc., Tokyo, Japan, clone H7507, dilution 1:1,000), mouse monoclonal anti human
estrogen receptor (ER; Dako, clone 1D5, dilution 1:50) and rabbit polyclonal anti human
progesterone receptor (PgR; Santa Cruz, Tokyo, Japan, clone C-19, dilution 1:1,000). After
rinsing with PBS, the sections were incubated with peroxidase-labelled polymer (Dako) for
60 min at room temperature and washed with PBS. Immunoreactivity was visualized with 0.05M
Tris buffer containing 3,3′-diaminobenzidine tetrachloride (DAB) and 0.027% hydrogen
peroxide (pH 7.5) for 2–10 min at room temperature. The sections were then washed with
Tris buffer and PBS, counter-stained for 20 s in Mayer’s hematoxylin, dehydrated, cleared
in xylene, and mounted.

IHC examination was quantified by counting immunostained nuclei as described previously
[14]. Three areas at magnification 400× per the
three elements (the acini, ducts and lobules consisted of oxyphilic cells) were selected
randomly. A percentage of the total epithelial cell nuclei examined was recognized as the
results of IHC examination. The ducts, acini and oxyphilic cells in each microscopic field
contained approximately 50–80, 70–330 and 120–580 epithelial cells, respectively.

TEM examination
The specimens for TEM were prepared to observe the ultrastructure of oxyphilic cells in
male rat mammary glands as previously described manner [25]. The mammary gland tissues were cut into approximately 1-mm3
pieces, fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer,
and post-fixed in 1% osmium tetroxide. After dehydration through a graded ethanol series,
the samples were embedded in resin consisted of methylnadic anhydride (MNA), Epok812,
dodecenly succinic anhydride (DDSA) and 2,4,6-tris (dimethylaminomethyl) phenol (DMP-30).
Ultrathin sections were prepared and stained with uranyl acetate and lead citrate, then
observed using TEM (H-7000KU, Hitachi, Tokyo, Japan).

Statistical analysis
The mean differences were analyzed by Student’s t-test. Data are
presented as mean ± SD. Dr. SPSS II program for Windows (SPSS Inc., Chicago, USA) was used
for statistical analysis. P values<0.05 were considered to be
statistically significant. Mean differences were analyzed by Student’s
t-test. Mean percentages of AR, ER or PgR-positive cells were evaluated
by the nonparametric Mann-Whitney test.

Results
Gross examination
Grossly, brown-colored mammary gland tissues were observed in all animals at 50–100 days
old, however, they were not observed in all animals at 35 days old or younger (Fig. 1Fig. 1. Gross examination. Right mammary glands in male rats at 14 (a, b), 21 (c, d), 35
(e), 50 (f, g), 75 (h, i) and 100 days old (j). Brown mammary glands were observed
at 50–100 days old (f–j).

).

Observation by stereoscopic microscope in whole mount specimens
There observed morphological changes with age in mammary glands of male rats (Table 1Table 1. Morphological changes of the mammary glands in male rats (whole mount
specimens)
Age (days)	Number of rats	Immature TEB	TEB	AB	Lobule	Duct	Number of TEBs	
14	5	+	−	+	−	++	8.0 ± 2.3	
21	5	++	−	+	−	++	16.2 ± 6.8a)	
35	5	−	++	++	+	++	98.0 ± 17.4b)	
50	5	−	+	+	++	++	21.0 ± 12.3c)	
75	5	−	−	±	+++ 	+	0.0d)	
100	5	−	−	±	+++	±	0.0d)	
Grade:−; negative, ±; very slight, +; slight, ++; moderate, +++; marked. a)
P<0.05: significantly different from 14 days. b)
P<0.01: significantly different from 21 days. c)
P<0.01: significantly different from 35 days. d)
P<0.05: significantly different from 50 days. TEB, terminal
end bud: AB, alveolar bud.

). At 14 and 21 days old (Figs. 2a
and bFig. 2. Whole mount specimen. Morphological changes of the mammary glands in male rats at
14 (a), 21 (b), 35 (c), 50 (d), 75 (e) and 100 days old (f). TEB: terminal end bud,
AB: alveolar bud, LOB: lobule, Du: duct. Scale bars, 500 µm.

), the mammary glands were consisted of ducts (dendritically branched main
structures), immature TEBs (slightly enlarged terminal buds on a duct) and alveolar buds
(ABs; structures with several projections branching from a duct). At 35 days old (Fig. 2c), TEBs (markedly enlarged terminal bud parts
on a duct, shaped like teardrop) were appreciably observed and lobules (more mature
clustered structures with multiple projections) also appeared. At 50–100 days old (Figs. 2d–f), the mammary glands predominantly
consisted of the lobules while ABs and TEBs tended to decrease with age. The density of
mammary glands was getting higher with age mainly because of remarkable development of the
lobules.

TEBs numbers were counted in the distal portions of right mammary glands (Table 1). TEBs were observed at 14–50 days old and
the number of TEBs was highest at 35 days old. The number of TEBs increased approximately
6-fold between 21 and 35 days old. At 75 and 100 days old, no TEBs were found.

Histological examination
The histological changes with age were summarized in Table 2Table 2. Histological changes of the mammary glands in male rats (H&E stain)
Age (days)	Number of rats	Morphology at a young age	Oxyphilic cell	
		
Immature TEB	TEB	AB	Lobule	Duct	AB	Lobule	Duct	
14	5	++	−	+	−	++	−	−	−	
21	5	++	−	+	−	++	−	−	−	
35	5	−	++	++	++	++	−	−	−	
50	5	−	±	±	+	+	+	++	+	
75	5	−	−	−	−	±	±	++	+	
100	5	−	−	−	−	±	±	+++	±	
Grade: −; negative, ±; very slight, +; slight, ++; moderate, +++; marked. TEB,
terminal end bud: AB, alveolar bud.

. The male mammary glands contained small epithelial cells with scanty
cytoplasm at 14–35 days old (Figs. 3a–cFig. 3. Histological examination of the mammary glands in male rats at 14 (a), 21 (b), 35
(c) and 50 days old (d-f) (H&E stain). At 50 days old, both structures with
morphology at a young age (d) and oxyphilic cells (e) were observed. (f) is the
enlarged figure of structure with oxyphilic cells. TEB: terminal end bud, AB:
alveolar bud, Du: duct. Scale bars, 100 µm (a–e), 50
µm (f).

). The mammary glands were composed of ducts, ABs and immature TEBs at 14 and 21
days old, and TEBs with significantly multilayered epithelium were found in addition to
the abovementioned two elements (ducts and ABs) at 35 days old. At 35 days old, all TEBs
were matured; no immature TEBs were observed. At 50 days old (Figs. 3d–f), oxyphilic cells with abundant eosinophilic cytoplasm
appeared in the mammary glands in addition to structure at a young age (14–35 days old).
No oxyphilic cells were observed at 35 days and younger. At 75 and 100 days old (Figs. 4a-fFig. 4. Histological examination of the mammary glands in male rats at 75 (a, b) and 100
days old (c–f) (H&E stain). (e) and (f) are the enlarged figures of a lobule
consisted of oxyphilic cells. Du (y): duct with morphology at a young age, Du (oxy):
duct consisted of oxyphilic cells, LOB (oxy): lobule consisted of oxyphilic cells,
Scale Bars, 100 µm (a–d), 50 µm (e), 25
µm (f).

), lobules and ducts consisted of oxyphilic cells were found, and the lumens became
clearly smaller in diameter. Only a few ducts which found at a young age were observed,
but the elements consisted of oxyphilic cells almost completely replaced the morphology at
a young age. The cytoplasm of oxyphilic cells was granular and had many fine vacuoles
(Figs. 4e–f).

Immunohistochemical examination
The changes of hormone receptor expression in the mammary glands with age were shown in
Table 3Table 3. Hormone receptor expression of the mammary glands in male rats
Age (days)	14	21	35	50	75	100	
Number of rats	5	5	5	5	5	5	
	
AR	Acini	0.0	0.0	0.0	11.8 ± 6.5a)	NE	NE	
Ducts	0.0	0.0	0.0	22.9 ± 9.8a)	NE	NE	
OCs	NA	NA	NA	50.1 ± 8.1	58.4 ± 6.2	71.3 ± 5.9b)c)	
	
ER	Acini	26.3 ± 2.3	29.1 ± 3.9	39.3 ± 8.3d)	31.1 ± 12.0	NE	NE	
Ducts	16.4 ± 3.7	24.3 ± 3.5e)	27.4 ± 7.9d)	22.9 ± 2.2d)	NE	NE	
OCs	NA	NA	NA	11.6 ± 2.0	2.7 ± 0.6b)	2.2 ± 0.7b)	
	
PgR	Acini	2.0 ± 0.8	1.7 ± 1.2	9.7 ± 3.6e)f)	12.6 ± 6.9e)g)	NE	NE	
Ducts	2.5 ± 0.5	1.9 ± 2.2	5.6 ± 1.6d)g)	17.4 ± 3.1a)e)f)	NE	NE	
OCs	NA	NA	NA	45.9 ± 11.5	4.4 ± 2.1	1.7 ± 1.2b)h)	
Results are expressed as proportion (%) of positive cells. Percentage values are
expressed as mean ± SD. a) P<0.01: significantly different from
35 days. b) P<0.01: significantly different from 50 days. c)
P<0.05: significantly different from 75 days. d)
P<0.05: significantly different from 14 days. e)
P<0.01: significantly different from 14 days. f)
P<0.01: significantly different from 21 days. g)
P<0.05: significantly different from 21 days. h)
P<0.01: significantly different from 75 days. AR, androgen
receptor: ER, estrogen receptor: PgR, progesterone receptor: OCs, oxyphilic cells:
NA, Not applicable: NE, Not evaluated.

. Representative pictures at 35 and 100 days old were shown in Fig. 5Fig. 5. Immunohistochemical examination of the mammary glands in male rats. In the mammary
gland at 35 days old (a, c, e), androgen receptor (AR) expression was not found (a)
while estrogen receptor (ER) expression was found (c, arrows). Progesterone receptor
(PgR) expression was observed in a small number of cells. The inset of (e) shows a
PgR positive cell (arrow). In the oxyphilic cells at 100 days old (b, d, f), the
portion of AR expression was high while the portions of ER and PgR were low. (a, b)
AR, (c, d) ER, (e, f) PgR. Scale Bars, 25 µm.

. In the mammary glands at 14–35 days old, AR expression was not found. At 50 days
old, AR expression in the mammary glands was seen in the acini, ducts and oxyphilic cells.
The proportion of AR expression in the oxyphilic cells increased with age from 50 days old
and was higher than those in acini and ducts.

ER and PgR expressions were found in the acini and ducts at 14–50 days old and in the
oxyphilic cells at 50–100 days old. The proportion of ER expression in epithelial cells of
acini and ducts increased with age at 14–35 days old but decreased at 50 days old. In
oxyphilic cells, the proportion of ER and PgR expressions was the highest at 50 days old
while that of ER expression was lower than those of AR and PgR expressions. In addition,
the proportions of ER and PgR expressions in the oxyphilic cells from 50 days old
decreased with age, and the decrease of PgR expression between 50 and 75 days old was
prominent. The proportions of ER and PgR expressions at 75 and 100 days old were extremely
lower comparing with the proportion of AR expression.

TEM examination
In TEM examination of oxyphilic cells, a number of lipid droplets were found in the
cytoplasm of oxyphilic cells. Lipid droplets were various in size. In addition, deformed
and/or enlarged mitochondria, lysosomes and peroxisomes were observed in the cytoplasm.
Small lumens with microvilli on their wall were also seen (Fig. 6Fig. 6. Transmission electron microscopic examination of oxyphilic cells of the mammary
glands in male rats at 75 days old. Small lumen with microvilli was found (b,
arrowhead). N: nucleus, G: Golgi apparatus, rER: rough-surfaced endoplasmic
reticulum, Ly: lysosome, P: peroxisome, M: mitochondria, Li: lipid drop. Scale bars,
1 µm.

).

Discussion
We investigated the change with age about mammary gland development of male rats and
evaluated the relation between the mammary gland morphological development and the hormone
receptor expression, focusing on the oxyphilic cells. In the present study, grossly brown
mammary glands were observed from 50 days old in male rats. Histologically, the epithelial
cells composing mammary glands at 14–35 days old were different from those from 50 days old.
The oxyphilic cells were observed from 50 days old, when the gross brown-colored change was
detected. From whole mount specimen observation, the developments of ABs and lobules from
the ducts and TEBs were seen with age. TEBs are solid or semisolid bulbous clusters of
immature epithelial cells at the ends of ducts and important sites of epithelial cell
proliferation during puberty [24]. The number of TEBs
showed a similar tendency to the reported data about the mammary glands of female rats
[8]. The number of TEBs prominently increased
between 21 and 35 days old; consistent with a previous study about male rats [8].

Previous studies have demonstrated that the mammary glands in adult rats showed sexual
dimorphism and male rats had male-specific cells [4,
8, 9, 11, 21, 22, 32, 33]. Sexual dimorphism in mammary glands has not been
reported in other mammalian animals including mice [4]. The male-specific cells were consistent with oxyphilic cells in our study. The
male-specific oxyphilic cells are considered to be derived from mammary epithelial and
ductal epithelial cells by histological examination and the previous study [28]; however, they are not poorly understood in its
development and ultrastructure. The TEM showed that the oxyphilic cells had numerous lipid
droplets in various sizes and cytoplasm contained deformed and/or enlarged mitochondria,
lysosomes and peroxisomes. Abundant cytoplasm might be caused by increased lipid droplets
and large size mitochondria. In canine mammary gland tumor, the cells exhibited oncocytic
metaplasia had abundant finely granular, eosinophilic and occasionally vacuolated cytoplasm,
resulted from abundant mitochondria [26]. It is
suggested that oxyphilic change in the male rat mammary glands is also related to enlarged
mitochondria. There were some limitations to study, and we couldn’t identify the biological
significance of oxyphilic cells. Further studies are required to confirm their role in male
rats.

In this study, the morphological changes with age are related to the expressions of sex
hormone receptors. ER expression was mainly observed in the mammary glands at 14–35 days
old, and AR expression was seen only from 50 days old, when oxyphilic cells appeared. Our
data confirm that AR expression was found in most oxyphilic cells, thus AR is considered to
be related to oxyphilic cells. Previous reports have demonstrated that the morphology of the
mammary glands in rats was affected by their hormonal conditions [5, 30, 33]. In the mammary glands of castrated young male rats (about 80 days
old) [33], the number of epithelial cells
significantly decreased and the alveoli lumen was more obvious when compared with that of
intact male rats. However, 5alpha-dihydrotestosterone administration returned their mammary
gland structure to that of the intact male rats. Conversely, the mammary glands in female
rats treated with androgen were similar to those in intact male rats [5, 30, 33]. Serum testosterone concentration in male rats significantly
increased from 4 days old and increased with ages at 21–160 days old, which is approximately
10 times higher than that in female rats at over 45 days old [1, 2]. In addition, sexual maturity in male
rats, preputial separation, is known to occur at approximately 45–48 days old [18]. In this study, the appearance and high AR expression
from 50 days old in the oxyphilic cells are considered to be associated with sexual maturity
and blood androgen increased period. Therefore, it suggests that higher androgen exposure
would be related to male-specific morphology of the mammary glands.

In contrast, estrogen is associated with normal mammary gland development in female rats,
especially with TEB formation in the peri-pubertal period [9, 16]. Male rats exposed to ethynyl
oestradiol during gestation and lactation had an increased number and density of TEBs
compared with control animals [23]. These suggest
that estrogen plays essential roles in mammary gland development in both male and female
rats. In this study, ER expression was mainly seen at 14–35 days old; therefore, the effect
of estrogen might regulate the mammary gland development during that period. However, blood
estrogen level was markedly lower in male rats than in female rats up to 21 days old [2]. In addition, there was no obvious structural
difference about mammary glands between wild-type and estrogen receptor knockout (ERKO) male
mice while mammary glands were undeveloped in ERKO female mice compared with wild type
female mice [3]. It is reasonably assumed that the
effect of estrogen via ER does not play the main role in the male mammary gland development.
We also examined PgR expression. Progesterone is known to be essential in ductular and
lobuloalveolar changes in prepubescent development and pregnancy [21]. Serum progesterone level had no clear difference between both sexes
from birth to 21 days old in rats [6]. In male rats at
100 days old, progesterone level was lower than in female rats [12]. In this study, the PgR expression was seen at from 14 to 100 days
old and the proportion of PgR expression markedly decreased from 75 days old. The
progesterone might be related to male mammary gland development at young ages, not at adult
ages. On the other hand, another report showed that no PgR expressions in mammary glands of
male rats were observed at from 1 to 70 days old [8].
This difference on the PgR expression from our study was considered due to the differences
of primary antibody used for IHC and rat strain. In addition, the other elements including
prolactin and growth hormone might be related to the mammary gland development in male rats,
similar to the report in female rats [29]. Further
studies are required to reveal the relationship of sex hormone and other elements including
prolactin and growth hormone with mammary gland development in male rats.

In conclusions, the oxyphilic cells appeared from 50 days old in mammary glands of male
rats and were associated with high expression of AR. In addition, their abundant
eosinophilic cytoplasm was suggested to be caused by an increased number of lipid droplets
and enlarged/deformed mitochondria.

Conflict of Interest
There were no conflicts of interest.

We are grateful to Mr. T. Kodama and Mr. M. Souda for their valuable technical assistance.
This research was partly supported by Japan Society for the Promotion of Science (JSPS)
KAKENHI [grant number 16K08023].
==== Refs
References
1 Banu S.K. Govindarajulu P. Aruldhas M.M.  2002 . Testosterone and estradiol up-regulate
androgen and estrogen receptors in immature and adult rat thyroid glands in
vivo . Steroids 67 :
1007 –1014 .
doi: 10.1016/S0039-128X(02)00063-6 12441185 
2 Banu S.K. Govindarajulu P. Aruldhas M.M.  2002 . Developmental profiles of TSH, sex
steroids, and their receptors in the thyroid and their relevance to thyroid growth in
immature rats .
Steroids 67 :
137 –144 . doi:
10.1016/S0039-128X(01)00144-1 11755178 
3 Bocchinfuso W.P. Hively W.P. Couse J.F. Varmus H.E. Korach K.S.  1999 . A mouse mammary tumor virus-Wnt-1
transgene induces mammary gland hyperplasia and tumorigenesis in mice lacking estrogen
receptor-alpha . Cancer
Res. 59 : 1869 –1876 .
10213494 
4 Cardy R.H.  1991 . Sexual dimorphism of the normal rat
mammary gland . Vet.
Pathol. 28 : 139 –145 .
doi: 10.1177/030098589102800206 1676552 
5 Chambô-Filho A. Camargos A.F. Pereira F.E.  2005 . Morphological changes induced by
testosterone in the mammary glands of female Wistar rats .
Braz. J. Med. Biol. Res. 38 :
553 –558 . doi:
10.1590/S0100-879X2005000400008 15962180 
6 Döhler K.D. Wuttke W.  1974 . Serum LH, FSH, prolactin and progesterone
from birth to puberty in female and male rats .
Endocrinology 94 :
1003 –1008 .
doi: 10.1210/endo-94-4-1003 4818764 
7 Ferlay J. Soerjomataram I. Dikshit R. Eser S. Mathers C. Rebelo M. Parkin D.M. Forman D. Bray F.  2015 . Cancer incidence and mortality worldwide:
sources, methods and major patterns in GLOBOCAN 2012 .
Int. J. Cancer 136 :
E359 –E386 .
doi: 10.1002/ijc.29210 25220842 
8 Filgo A.J. Foley J.F. Puvanesarajah S. Borde A.R. Midkiff B.R. Reed C.E. Chappell V.A. Alexander L.B. Borde P.R. Troester M.A. Bouknight S.A. Fenton S.E.  2016 . Mammary Gland Evaluation in Juvenile
Toxicity Studies: Temporal Developmental Patterns in the Male and Female Harlan
Sprague-Dawley Rat . Toxicol.
Pathol. 44 : 1034 –1058 .
doi: 10.1177/0192623316663864 27613106 
9 Funato M. Kawaguchi H. Hori T. Yoshikawa T. Gejima K. Kawashima H. Taguchi S. Ninomiya K. Umekita Y. Nagata R. Yoshida H.  2006 . Effects of neonatally-administered
17beta-estradiol on induction of mammary carcinomas by 7, 12-dimethylbenz[a]anthracene
in female rats . In
Vivo 20 : 33 –37 . 16433025 
10 Giordano S.H. Cohen D.S. Buzdar A.U. Perkins G. Hortobagyi G.N.  2004 . Breast carcinoma in men: a
population-based study .
Cancer 101 :
51 –57 . doi:
10.1002/cncr.20312 15221988 
11 Gouda Z.A. Ahmed S.M. Elghonaimy N.M.  2015 
Characterization of adult male albino rat
mammary gland: a histological and serological study . J.
Histol. Histopathol. 2 : 5 . doi:
10.7243/2055-091X-2-5 
12 Goya R.G. Lu J.K. Meites J.  1990 . Gonadal function in aging rats and its
relation to pituitary and mammary pathology . Mech.
Ageing Dev. 56 : 77 –88 .
doi: 10.1016/0047-6374(90)90116-W 2259256 
13 Kawaguchi H. Miyoshi N. Miyamoto Y. Souda M. Umekita Y. Yasuda N. Yoshida H.  2010 . Effects of fetal exposure to
4-n-octylphenol on mammary tumorigenesis in rats . In
Vivo 24 : 463 –470 .
20668312 
14 Kawaguchi H. Umekita Y. Fukuzaki K. Maeda H. Miyajima H. Nagata R. Yoshida H.  2009 . Expression of androgen receptor in
mammary glands in ovariectomized cynomolgus monkeys .
Vet. Pathol. 46 :
526 –530 . doi:
10.1354/vp.08-VP-0134-K-AM 19176497 
15 Kawaguchi H. Umekita Y. Souda M. Gejima K. Kawashima H. Maeda H. Miyajima H. Nagata R. Yoshida H.  2007 . Increased bone mineral density in aged
rats with spontaneous mammary dysplasia . In
Vivo 21 : 583 –586 .
17708349 
16 Kleinberg D.L. Ruan W.  2008 . IGF-I, GH, and sex steroid effects in
normal mammary gland development . J. Mammary Gland Biol.
Neoplasia 13 : 353 –360 .
doi: 10.1007/s10911-008-9103-7 19034633 
17 Korde L.A. Zujewski J.A. Kamin L. Giordano S. Domchek S. Anderson W.F. Bartlett J.M. Gelmon K. Nahleh Z. Bergh J. Cutuli B. Pruneri G. McCaskill-Stevens W. Gralow J. Hortobagyi G. Cardoso F.  2010 . Multidisciplinary meeting on male breast
cancer: summary and research recommendations . J. Clin.
Oncol. 28 : 2114 –2122 .
doi: 10.1200/JCO.2009.25.5729 20308661 
18 Lewis E.M. Barnett J.F. JrFreshwater L. Hoberman A.M. Christian M.S.  2002 . Sexual maturation data for Crl
Sprague-Dawley rats: criteria and confounding factors .
Drug Chem. Toxicol. 25 :
437 –458 . doi:
10.1081/DCT-120014794 12378951 
19 Li S. Han B. Liu G. Li S. Ouellet J. Labrie F. Pelletier G.  2010 . Immunocytochemical localization of sex
steroid hormone receptors in normal human mammary gland .
J. Histochem. Cytochem. 58 :
509 –515 . doi:
10.1369/jhc.2009.954644 20026671 
20 Líška J. Brtko J. Dubovický M. Macejová D. Kissová V. Polák Š. Ujházy E.  2016 . Relationship between histology,
development and tumorigenesis of mammary gland in female rat .
Exp. Anim. 65 :
1 –9 . doi:
10.1538/expanim.15-0055 26424555 
21 Lucas J.N. Rudmann D.G. Credille K.M. Irizarry A.R. Peter A. Snyder P.W.  2007 . The rat mammary gland: morphologic
changes as an indicator of systemic hormonal perturbations induced by
xenobiotics . Toxicol.
Pathol. 35 : 199 –207 .
doi: 10.1080/01926230601156260 17366314 
22 Mandrup K. Boberg J. Isling L.K. Christiansen S. Hass U.  2016 . Low-dose effects of bisphenol A on
mammary gland development in rats .
Andrology 4 :
673 –683 . doi:
10.1111/andr.12193 27088260 
23 Mandrup K.R. Hass U. Christiansen S. Boberg J.  2012 . Perinatal ethinyl oestradiol alters
mammary gland development in male and female Wistar rats .
Int. J. Androl. 35 :
385 –396 . doi:
10.1111/j.1365-2605.2012.01258.x 22428746 
24 Masso-Welch P.A. Darcy K.M. Stangle-Castor N.C. Ip M.M.  2000 . A developmental atlas of rat mammary
gland histology . J. Mammary Gland Biol.
Neoplasia 5 : 165 –185 .
doi: 10.1023/A:1026491221687 11149571 
25 Matsumoto M. Kurohmaru M. Hayashi Y. Nishinakagawa H. Otsuka J.  1994 . Permeability of mammary gland capillaries
to ferritin in mice . J. Vet. Med.
Sci. 56 : 65 –70 . doi: 10.1292/jvms.56.65 8204762 
26 Nagahara R. Kimura M. Itahashi M. Sugahara G. Kawashima M. Murayama H. Yoshida T. Shibutani M.  2016 . Canine mammary minute oncocytomas with
neuroendocrine differentiation associated with multifocal acinar cell oncocytic
metaplasia . J. Vet. Diagn.
Invest. 28 : 722 –728 .
doi: 10.1177/1040638716664381 27698175 
27 Ninomiya K. Kawaguchi H. Souda M. Taguchi S. Funato M. Umekita Y. Yoshida H.  2007 . Effects of neonatally administered
diethylstilbestrol on induction of mammary carcinomas induced by 7,
12-dimethylbenz(a)anthracene in female rats . Toxicol.
Pathol. 35 : 813 –818 .
doi: 10.1080/01926230701584205 17943655 
28 Rudmann D.G. Cohen I.R. Robbins M.R. Coutant D.E. Henck J.W.  2005 . Androgen dependent mammary gland virilism
in rats given the selective estrogen receptor modulator LY2066948
hydrochloride . Toxicol.
Pathol. 33 : 711 –719 .
doi: 10.1080/01926230500343902 16263696 
29 Russo I.H. Twari M. Russo J.  1989 . Morphology and development of the rat mammary gland. pp.
304–314. In: Monographs on Pathology of Laboratory Animals: Integument and Mammary Glands
(Jones T. C., Mohr U., and Hunt R. D., eds.), Springer Verlag, Berlin.
30 Sourla A. Martel C. Labrie C. Labrie F.  1998 . Almost exclusive androgenic action of
dehydroepiandrosterone in the rat mammary gland .
Endocrinology 139 :
753 –764 . doi:
10.1210/endo.139.2.5762 9449650 
31 Umekita Y. Souda M. Hatanaka K. Hamada T. Yoshioka T. Kawaguchi H. Tanimoto A.  2011 . Gene expression profile of terminal end
buds in rat mammary glands exposed to diethylstilbestrol in neonatal
period . Toxicol.
Lett. 205 : 15 –25 .
doi: 10.1016/j.toxlet.2011.04.031 21658437 
32 Wang X.J. Bartolucci-Page E. Fenton S.E. You L.  2006 . Altered mammary gland development in male
rats exposed to genistein and methoxychlor . Toxicol.
Sci. 91 : 93 –103 .
doi: 10.1093/toxsci/kfj120 16443925 
33 Watson P.A. Kim K. Chen K.S. Gould M.N.  2002 . Androgen-dependent mammary carcinogenesis
in rats transgenic for the Neu proto-oncogene . Cancer
Cell 2 : 67 –79 . doi: 10.1016/S1535-6108(02)00083-1 12150826 
34 White J. Kearins O. Dodwell D. Horgan K. Hanby A.M. Speirs V.  2011 . Male breast carcinoma: increased
awareness needed . Breast Cancer
Res. 13 : 219 . doi: 10.1186/bcr2930 22017761 
35 Yoshida H. Kadota A. Fukunishi R.  1980 . Pathology of early lesions of mammary
carcinoma and mammary dysplasia induced by 7,12-dimethylbenz(a)anthracene in the
neonatally androgenized Sprague-Dawley female rats .
Virchows Arch. B Cell Pathol. Incl. Mol.
Pathol. 34 : 33 –41 .
doi: 10.1007/BF02892405 6108005 
36 Yoshikawa T. Kawaguchi H. Umekita Y. Souda M. Gejima K. Kawashima H. Nagata R. Yoshida H.  2008 . Effects of neonatally administered
low-dose diethylstilbestrol on the induction of mammary carcinomas and dysplasias
induced by 7,12-dimethylbenz [a] anthracene in female rats .
In Vivo 22 :
207 –213 . 18468405