BIOLOGY OF REPRODUCTION 84, 1094–1102 (2011)
Published online before print 26 January 2011.
Activation of the Aryl Hydrocarbon Receptor During Pregnancy in the Mouse Alters
Mammary Development Through Direct Effects on Stromal and Epithelial Tissues1
Betina J. Lew,3Ravikumar Manickam, and B. Paige Lawrence2
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry,
Rochester, New York
Activation of the aryl hydrocarbon receptor (AHR), an
environment-sensing transcription factor, causes profound im-
pairment of mammary gland differentiation during pregnancy.
Defects include decreased ductal branching, poorly formed
alveolar structures, suppressed expression of milk proteins, and
failure to nutritionally support offspring. AHR is activated by
numerous environmental toxins, such as 2,3,7,8-tetrachlorodi-
benzo-p-dioxin (TCDD), and plays an as yet poorly understood
role in development and reproduction. To better understand
how AHR activation affects pregnancy-associated mammary
gland differentiation, we used a combination of ex vivo
differentiation, mammary epithelial transplantation, and AHR-
deficient mice to determine whether AHR modulates mammary
development through a direct effect on mammary epithelial cells
(MECs) or by altering paracrine or systemic factors that drive
pregnancy-associated differentiation. Studies using mutant mice
that express an AHR protein lacking the DNA-binding domain
show that defects in pregnancy-associated differentiation re-
quire AHR:DNA interactions. We then used fluorescence-based
cell sorting to compare changes in gene expression in MECs and
whole mammary tissue to gain insight into affected signaling
pathways. Our data indicate that activation of the AHR during
pregnancy directly affects mammary tissue development via
both a direct effect on MECs and through changes in cells of the
fat pad, and point to gene targets in MECs and stromal tissues as
putative AHR targets.
agalactia, alveolarization, environment, environmental factors,
lactation, lactogenesis, mammary glands, TCDD, toxicology
The mammary gland develops mostly during postnatal life.
At birth, it is composed of a simple network of ducts and
terminal end buds (TEBs), which are highly proliferative
immature structures. With the onset of puberty, under the
control of reproductive hormones, the TEBs proliferate and
differentiate, and secondary and tertiary ducts invade the fat
pad, after which glandular development is minimal . During
pregnancy, the gland resumes its growth, with a highly
proliferative phase in the beginning of pregnancy, when ductal
branching takes place, followed by a phase in which epithelial
cells differentiate into lobule-alveolar structures that will
eventually produce milk [2, 3]. This complex process of
postnatal mammary development is coordinated by numerous
molecules that are produced both systemically, in several
glands and tissues, and locally by the cells of the fat pad
(stroma) and mammary epithelium [4–6]. Extrinsic factors such
as nutrition and anthropogenic chemicals affect and potentially
deregulate this well-orchestrated process [7–12]. One target
pathway for exogenous chemicals to influence mammary gland
development is via activation of the aryl hydrocarbon receptor
(AHR). Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), which is an AHR-specific agonist and common
environmental pollutant, causes severe defects in mammary
gland development . During pregnancy, injuries to the
gland include decreased epithelial cell proliferation in early
pregnancy and midpregnancy, stunted longitudinal growth,
decreased ductal branching, poor formation of alveolar
structures, and reduced expression of milk proteins. Collec-
tively, these defects in development result in an inability to
nutritionally support offspring [9, 12].
The AHR is a member of the per-arnt-sim (PAS) family of
basic-region-helix-loop-helix (bHLH) transcription factors and
directs the expression of many detoxification genes . Upon
activation by ligands such as TCDD, the AHR translocates to
the nucleus and induces the expression of genes for several
metabolic enzymes. Thus, one role of AHR is to provide
protection from toxic chemicals by stimulating expression of
enzymes to eliminate them. In addition to promoting
metabolism and clearance of xenobiotics, recent studies
suggest that AHR modulates physiological processes and
molecular cascades that regulate proliferation, differentiation,
and apoptosis [14–17]. Moreover, phenotypic differences such
as decreased liver development, decreased body size over the
first 4 wk of age, altered heart, ovary, and other organ
development, and increased mortality rate were noted in AHR
knockout (AhrKO) mice when compared to wild-type (WT)
littermates [18–21], further emphasizing that there is a normal,
physiological role for AHR.
Although it has not been thoroughly evaluated, data
regarding the AHR’s role in mammary gland development
present a mixed picture, but collectively they suggest a possible
endogenous role of the AHR in pregnancy-associated mam-
mary development [22–24]. Moreover it is clear that AHR
activation by exogenous ligands causes severe defects in
pregnancy-associated mammary gland growth and lactogenesis
[9, 12]. Understanding the precise molecular mechanisms that
control mammary gland development during pregnancy
remains an active area of research. Work from numerous
laboratories has shown that specific factors regulate distinct
phases of this process; thus, there are many potential molecular
1Supported by research and training grants from the National Institutes
of Health (K02-ES012409, R21-ES013863, R01-ES013958 to B.P.L.),
T32-ES07026, and Environmental Health Sciences Center (P30-
ES01247) Pilot Project Award (to B.J.L. and B.P.L.). B.J.L. is the
recipient of a Seed the Scientist Award from the Art BeCAUSE
2Correspondence: B. Paige Lawrence, Box 850, University of Rochester
Medical Center, Rochester, NY 14642. FAX: 585 276 0239;
3Current address: Procter and Gamble, Cincinnati, OH.
Received: 30 July 2010.
First decision: 2 September 2010.
Accepted: 21 January 2011.
? 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
Downloaded from www.biolreprod.org.
targets of AHR [4, 25–27]. We have previously reported that
TCDD does not cause defects in lactogenic differentiation by
simply altering plasma levels of prolactin, estradiol, or
progesterone . However, in addition to circulating hormone
levels, development of the mammary gland is controlled by
many other molecules that are produced locally by the cells of
the mammary fat pad and epithelium, and systemically by other
organs, such as the liver [2, 4, 26, 27]. Failed lactogenesis
could therefore result from AHR-mediated direct effects on
mammary gland tissues or indirectly by disrupting levels of
systemically derived regulatory factors. Furthermore, within
the mammary gland itself, molecules derived from the stroma
(e.g., fat pad) and parenchymal tissues (e.g., mammary
epithelial cells) regulate the process of lactogenesis, and
TCDD exposure could perturb events within either or both of
The objectives of the present study were to determine
whether: 1) the effects of TCDD on pregnancy-associated
mammary development are mediated by the AHR and if the
loss of AHR’s DNA-binding domain alters the normal
pregnancy-associated mammary development, (2) exposure to
TCDD during pregnancy alters mammary development
through a direct effect on the mammary tissue, and (3) defects
in mammary differentiation and lactogenesis result from direct
effects on mammary epithelial cells (MECs). Determining
whether AHR activation by TCDD affects the normal course of
mammary development by altering processes that occur locally
in the mammary gland and within the fat pad and/or the
epithelium is critical for identifying the underlying molecular
targets that may be injured by exposure to exogenous
chemicals during pregnancy.
MATERIALS AND METHODS
Animals and Treatment
All the animal treatments were conducted with the approval of the
Institutional Animal Care and Use Committee of the University of Rochester
School of Medicine and Dentistry. Animals were housed in microisolator units,
had access to food and water ad libitum, and were maintained on a 12L:12D
cycle. Wild-type C57BL/6 (Ahrb) mice were either bred in-house or purchased
(National Cancer Institute, Frederick, MD). A breeding stock of AHR-deficient
mice (B6.129-Ahrtm1G8nz, AhrKO), was obtained from the National Cancer
Institute . A breeding stock of Ahrdbd/dbdmice was generously provided by
Dr. Christopher Bradfield (University of Wisconsin, Madison, WI). Ahrdbd/dbd
mice carry a deletion of the DNA binding domain in the Ahr gene such that
they express AHR protein that can bind ligand and migrate to the nucleus, but
cannot bind to DNA . Although Ahrdbd/dbdmice have been backcrossed
onto a C57BL/6 background, the Ahrdbd/dbdlineage carries the Ahrdallele. This
allele encodes a protein with 10-fold lower binding affinity for TCDD; thus,
mice on an Ahrdallele background require a 10-fold higher dose of TCDD than
the Ahrballele WT mice in order to elicit similar effects . For some
experiments, heterozygous (Ahrþ/?) mice were bred to produce age-matched
homozygous null (AhrKO) and WT offspring. Colonies of B6.Ahrd, AhrKO,
and Ahrdbd/dbdmutant mice are maintained in-house. Female offspring were
genotyped at 15 days of age using DNA extracted from an ear punch (QIAamp
DNA mini kit; Qiagen, Valencia, CA). PCR was carried out with Gene Amp
XL PCR kit (Applied Biosystems, Foster City, CA) using AHR sense and
antisense primers according to previous protocol for genotyping AhrKO 
and Ahrdbd/dbdmice .
For timed-pregnant mice, virgin females were placed with males and
checked daily for a vaginal plug. The day of the vaginal plug was considered
the day of pregnancy 0 (DP 0), and females were then housed individually for
the duration of the experiment. In order to analyze the development of
mammary glands of age-matched WT, AhrKO, and Ahrdbd/dbdmice, animals
were mated between 9 and 11 wk of age and tissue was collected on the day of
TCDD (?98% purity; Cambridge Isotopes Laboratory, Andover, MA) was
dissolved in 0.1% anisole and diluted in peanut oil. The vehicle control consisted
of an equivalent concentration of anisole in peanut oil. Impregnated animals
were gavaged with 5 lg/kg or 50 lg/kg body weight of TCDD (Ahrband Ahrd
allele mice, respectively) or peanut oil vehicle control. The half-life of TCDD in
C57BL/6 mice is approximately 8–10 days ; thus, in order to maintain AHR
activation throughout pregnancy, TCDD was administered every 7 days (i.e., on
DP 0, DP 7, and DP 14). Animals were killed on DP 6 or on the day of
parturition, and mammary glands were removed for analyses. The administered
dose of 5 lg/kg of TCDD to mice with the b-allele of the AHR is well below the
LD50of 296 lg/kg determined for C57BL/6 mice . Moreover, this dose
range (5–20 lg/kg) is widely used in mouse studies designed to understand the
mechanisms by which AHR affects in vivo physiology.
Mammary Gland Transplantation
Transplantation of the mammary epithelium was carried out as previously
described . Briefly, female AhrKO and WT mice (19–21 days old) were
anesthetized with isofluorane. An L-shape small incision was made between the
fourth and fifth nipple, and the epithelial tissue from the fourth abdominal right
gland from each mouse was identified and removed. The newly cleared fat pad
of the AhrKO mouse was then immediately implanted with a small piece (0.5–1
mm diameter) of the WT epithelium, and vice versa. Six to eight wk after
surgery (9–11 wk of age), transplant recipients were housed with males for
impregnation and exposure to TCDD or vehicle (see Fig. 4A).
Ex Vivo Mammary Gland Development
Female C57BL/6 virgin mice (23–25 days old) were implanted subcuta-
neously with pellets containing a mixture of estrogen, progesterone, and
cholesterol at a ratio of 1:1000:2002 (Innovative Research of America,
Sarasota, FL). Fifteen days later, the abdominal and thoracic mammary glands
were collected and incubated as previously described [35, 36]. Glands remained
floating on the top of a siliconized lens-cleaning tissue (105; Whatman,
Maidstone, U.K.) in Waymouth medium containing 100 IU/ml penicillin and
100 lg/ml streptomycin (Gibco Invitrogen Cell Culture, Carlsbad, CA), 20 mM
Hepes, and a lactogenic mix (LM) containing the following: bovine insulin (5
lg/ml), aldosterone (100 ng/ml), hydrocortisone (100 ng/ml), mouse epidermal
growth factor (60 ng/ml), sheep prolactin (1 lg/ml), and 0.1% bovine serum
albumin. All the proteins and hormones used in the LM were obtained from
Sigma-Aldrich (St. Louis, MO). In addition to LM, glands were exposed to
either 10 nM TCDD diluted in dimethyl sulfoxide (DMSO) or 0.1% DMSO as
vehicle control. Right thoracic glands served as controls for preincubation
development. Following 6 days in culture, whole mounts were prepared from
the left abdominal glands and analyzed for morphological development. The
right abdominal glands were stored at ?808C for protein analysis.
Morphological Development Analyses
Mammary gland whole mounts were prepared as described previously [9,
12]. Briefly, evaluation of mammary development was performed without
knowledge of treatment by at least two different scientists. Glands were given a
developmental score based on a four-point scale (1 ¼ poor development/
differentiation to 4 ¼ excellent growth and development). The subjective
scoring scales were specific to the stage of development. For glands collected
on the day of parturition, development was evaluated according to the quality
and quantity of lobuloalveolar units. In addition to a subjective scoring, the
assessment of the tissue obtained from ex vivo incubation was performed based
on the number and quality of lobuloalveolar and branching morphogenesis. The
quantitative morphometric analysis of postincubation tissues was made in two
sections that were randomly selected from different regions of photographed
mammary gland whole mounts. Digital micrographs were evaluated in a
blinded mode. The number of lobuloalveolar units was determined using
printed images representing 1.56 mm2of tissue. The number of branches in a
total linear length of about 6 mm of duct was evaluated using a PlanWheel SA2
(Scalex Corporation, Carlsbad, CA). Mean scores for each group were
computed and analyzed for differences due to treatment.
Mammary tissue was homogenized in buffer containing 10 mM Hepes, 1
mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 0.6% NP-40, 0.1
M phenylmethylsulfonyl fluoride, 10 lg/ml aprotinin, and 10 lg/ml leupeptin.
Protein concentration was determined, and 25 lg of protein from each sample
was subjected to SDS-PAGE and then transferred to nitrocellulose membranes.
Beta-casein protein was visualized by probing with a goat polyclonal anti-mouse
b-casein antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by
horseradish peroxidase-conjugated donkey anti-goat antibody (Santa Cruz
Biotechnology, Inc.). Antibodies to b-actin (Sigma Chemical Co., St. Louis,
MO) were used as a control. Antibody complexes were visualized using
enhanced chemiluminescent reagents (Amersham Pharmacia, Piscataway, NJ).
TCDD AND PREGNANCY-ASSOCIATED MAMMARY DEVELOPMENT
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Single Cell Suspensions from Mammary Glands
To isolate MECs, we pooled the second and third thoracic and the fourth
abdominal glands of three animals per sample; the glands were collected in
such a way that lymph nodes were excised prior to preparing single cell
suspensions, which was done as previously described . Briefly, dissociation
was performed by mincing the tissue in DMEM/F12 media containing 5% fetal
bovine serum (FBS) (Gibco Invitrogen), 300 U/ml collagenase, and 100 U/ml
hyaluronidase mix (StemCell Technologies, Vancouver, Canada), followed by
2-h incubation on a rocking tray at 378C. Red blood cells were eliminated using
an ammonium chloride lysing solution (0.15 M NH4Cl, 10 mM NaHCO3, 1
mM EDTA). A single cell suspension was then obtained by sequential
enzymatic treatments with 0.25% trypsin-EDTA (Gibco Invitrogen) followed
by 5 mg/ml dispase II (StemCell Technologies) and 0.1 mg/ml DNase I
(StemCell Technologies). All the enzymatic reactions were interrupted by the
addition of cold Hanks balanced salt solution modified (HBSS) supplemented
with 2% FBS and filtered through a 40-lm cell strainer. After the final
centrifugation, cells were resuspended in 1 ml HBSS, and the number of cells
was determined using a Coulter Counter (Beckman Coulter, Brea, CA).
Fluorescence-Activated Cell Sorting
Single cell suspensions were incubated in a solution containing 50 lg/ml rat
immunoglobulin G and 5 lg/ml of anti-mouse CD16/32 antibody for 15 min
prior to staining with antibodies. The following antibodies were used: anti-
mouse CD24 (clone M1/69) conjugated to PerCP-Cy5.5 was used as a marker
for epithelial cells and a hematopoietic lineage panel containing biotinylated
anti-mouse CD3 (clone 145–2C11), CD45/B220 (clone RA3-6B2), CD11B
(clone M1/70), erythroid marker (clone TER-119), LY6G (clone RB6-8c5), and
CD31 (PECAM1, clone 390, 13–031) was used to exclude nonepithelial cells.
Fluorescein isothiocyanate-conjugated streptavidin was added to label
biotinylated antibodies. All the antibodies were purchased from eBioscience
(San Diego, CA). Dead cells were identified by the addition of 40,6-diamidino-
2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). DAPI?Lin?CD24þcells
were sorted using a FACSAria (BD, Franklin Lanes, NJ) and used for RNA
extraction (see Fig. 5).
RNA Extraction and Real-Time PCR
RNA was isolated and purified from whole tissue with Ribopure kit
(Applied Biosystems/Ambion, Austin, TX) and from isolated MECs with
RNAqueous (Applied Biosystems/Ambion) following the instructions provided
with the kits. When the abdominal glands were used, lymph nodes were excised
and not used in the tissue preparation or extraction of nucleic acids. Total RNA
was quantified using a nanodrop (Thermo Scientific, Wilmington, DE), and the
reverse transcriptase (RT) reaction was performed with oligo dT, followed by
enzymatic reaction with M-MLVRTase (Applied Biosystems/Ambion). Real-
time PCR was performed for Cyp1a1 (Forward: TTT GGA GCT GGG TTT
GAC AC; Reverse: CTG CCA ATC ACT GTG TCT A) (IDT Technologies,
Coralville, IA), insulin-like growth factor 1 (size band: 98, reference position
1322; SABiosciences, Frederick, MD), E-cadherin (size band: 186, reference
position: 3783; SABiosciences), cyclin D1 (size band: 172 reference position:
3249; SABiosciences), and SYBR green supermix (Biorad, Hercules, CA).
Amplification was performed on an iCycler MyiQ2 (Biorad) using triplicate
reactions. GAPDH and L13 were used as internal control reference genes, and
differences in gene expression were calculated using the DDCtmethod .
Data were analyzed using StatView software (SAS Software, Cary, NC).
Using a two-way ANOVA followed by posthoc tests (Bonferroni/Dunn test),
differences between independent variables were compared over time and
between treatment groups. Differences between two groups at a single point in
time were evaluated using a Student t-test. Differences were considered
significant when P values were ,0.05.
TCDD Alters Mammary Gland Development Through
Activation of the AHR and Its DNA-Binding Domain
The present data demonstrate that both AHR protein and its
DNA-binding domain are required for the deleterious effects
development are mediated through the
AHR and require its DNA-binding domain.
Nulliparous wild-type (WT), homozygous
Ahr?/?(AhrKO), or AhrKOdbd/dbdmice (age
9–11 wk) were impregnated and dosed with
TCDD as described in Materials and Meth-
ods. All the animals were killed on the day
of parturition, tissue was collected, and
whole mounts were prepared for the eval-
uation of development. Representative
whole mounts of mammary glands from (A)
vehicle and TCDD-treated AhrKO and WTb
(b-allele of Ahr), and (C) AhrKOdbd/dbdand
WTd(d-allele) show pregnancy-induced
differentiation. Representative images were
obtained using a Zeiss dissecting micro-
scope (3.13 magnification) with a Nikon
Coolpix E995 camera. B, D) Average
developmental scores were determined.
Error bars indicate the SEM, and asterisks
indicate statistically significant differences
between vehicle- and TCDD-treated mice
of the same genotype (P , 0.05, n ¼ 2–12).
Data are representative of two separate
The effects of TCDD on mammary
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on pregnancy-induced mammary gland development caused by
exposure to TCDD. However, neither is required for the normal
development of the mammary gland. As shown in Figure 1,
pregnant WT animals exposed to TCDD demonstrated a
profound defect in pregnancy-associated mammary develop-
ment. When compared to the vehicle treatment group,
mammary glands collected from TCDD-treated WT animals
had stunted growth; alveolar structures appeared poorly formed
and empty. Scoring of glandular development revealed scores
that were at least 50% lower than the vehicle-treated group
(Fig. 1, A–C). These changes correlated with a substantially
reduced expression of b-casein (data not shown). In contrast,
when TCDD was administered to AhrKO or Ahrdbd/dbdmice
(Fig. 1, A and B, respectively), none of the above defects were
noted. In addition, compared to mammary glands collected
from vehicle-treated WT animals, mammary glands from
AhrKO and Ahrdbd/dbdmice appeared to have normal
development during pregnancy, that is, on the day of
parturition, mammary glands from AhrKO and Ahrdbd/dbdmice
had alveolar structures that were completely formed, filled with
milk, and fully populated the fat pad. Moreover, these mice
were able to nutritionally support their offspring. Thus, AHR
does not appear to be required for the normal development of
mammary glands during pregnancy, but the deleterious effects
of TCDD on tissue growth and differentiation are AHR-
mediated and require AHR binding to DNA.
TCDD Exposure Alters Expression of Genes
on Mammary Tissue
Given that AHR is a transcription factor, that mammary
tissue expresses AHR [39–41], and that we have previously
reported that TCDD treatment reduces MEC proliferation and
differentiation , we examined whether AHR activation alters
the expression of genes related to cell cycle progression and
differentiation in early pregnancy (DP 6). While the expression
of many genes was unaffected by TCDD treatment, when
compared to tissue collected from animals treated with the
peanut oil vehicle, AHR activation by TCDD exposure induced
a 12-fold increase in the expression of the well-characterized
AHR target gene Cyp1a1 (Fig. 2A). In contrast, cyclin-D1 and
E-cadherin expression levels were reduced 2- to 3-fold in
glands from TCDD-exposed animals (Fig. 2, B and C,
respectively). These alterations in gene expression suggest that
AHR activation by TCDD impairs lactogenic differentiation, at
least in part, by altering the production of molecules involved
in tissue growth. However, molecules produced within the
mammary gland and signals derived from other organs control
expression of these factors; thus, these changes could be the
result of primary AHR targets in the endocrine system,
mammary fat pad, or in the mammary epithelium.
TCDD Impairs Mammary Tissue Development
by Direct Effects
To determine whether exposure to TCDD affects pregnan-
cy-associated mammary development by acting directly on the
mammary gland, we isolated glands from virgin mice and
induced differentiation of the whole organs in culture .
When mammary tissue was incubated with lactogenic stimuli,
the glands developed clearly noticeable secondary and tertiary
branching, the majority of the stromal tissue was covered by
lobule-alveoli structures (Fig. 3, A and B), and b-casein
expression was induced (Fig. 3E, left side). However, when
TCDD was added to the cultures, glandular development was
notably stunted (Fig. 3B). Secondary and tertiary branches
could be observed, but there were on average about 40% fewer
lobule-alveoli structures than in the control group (Fig. 3C). In
addition, the levels of b-casein were significantly decreased in
glands treated with TCDD (Fig. 3, D and E). These results
indicate that TCDD impairs mammary gland development
through direct action on mammary tissue. However, this does
mammary tissue. Bar graphs represent average (6SEM) level of gene
expression in mammary glands collected from WT animals exposed to 5
lg/kg TCDD or vehicle (Veh) on DP 0 and killed on DP 6. Average levels
of gene expression were calculated using the DDCtmethod and
normalized against L13 and vehicle control group. The graphs represent
the relative expression of the following genes: (A) Cyp1a1, (B) cyclin D1,
and (C) E-cadherin. Asterisks indicate statistically significant differences (P
, 0.05, n ¼ 6).
AHR activation during pregnancy alters gene expression in
TCDD AND PREGNANCY-ASSOCIATED MAMMARY DEVELOPMENT
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not rule out the possibility of additional contributions on
pregnancy-associated glandular differentiation due to AHR-
mediated actions on other organs and tissues.
Impaired Development Is a Consequence of Direct
and Indirect Effects on the Mammary Epithelium
We next sought to determine whether activation of the AHR
causes these changes through a direct or indirect impact on the
mammary epithelium. To accomplish this, we reciprocally
transplanted AhrKO epithelium into cleared fat pads of WT
animals, and vice versa, to create WT mice with AhrKO
epithelium and AhrKO animals with WT epithelium. There-
fore, in the WT recipient animals, all the organs and the
mammary stroma are responsive to TCDD, but the transplanted
epithelium is nonresponsive (AhrKO, Fig. 4C). On the other
hand, in AhrKO recipients all the organs and glands, including
the mammary fat pad tissue, are nonresponsive; however, the
transplanted mammary epithelium is responsive to TCDD
(WT, Fig. 4B). After recovery from surgery, transplant
recipients were impregnated and exposed to TCDD or vehicle
control, and mammary glands were collected on the day of
(3.23 magnification) from glands removed from the following groups: (A) virgin (25–28 days old) animals before culture and (B) after culture for 6 days
with LM containing 0.1% DMSO (Veh) or TCDD (10 nM). C) Bars represent the average number of alveolar structures on mammary glands exposed in
culture to 0.1% DMSO (Veh) or TCDD (10 nM). D) Immunoblots show levels of b-casein, a marker of mammary gland differentiation, in tissue
homogenates from WTanimals. Immunoblots were also probed with an antibody to b-actin, which served as a loading control. Each lane represents tissue
from a different animal. E) The bar graphs depict results of densitometric analysis of the average b-casein levels. Tissue collected from nonprimed animals
served as control for ex vivo differentiation, and they did not develop under any condition (data not shown). In addition, we cultured mammary glands
collected from AhrKO mice. Lack of AHR did not alter tissue development in this system, and ex vivo cultured AhrKO glands were not affected by TCDD
treatment (data not shown). Data represent mean 6 SEM (n ¼ 9; P , 0.05 compared to vehicle control). Results are representative of at least two
Ex vivo TCDD exposure impairs mammary development by direct effects on the mammary tissue. Representative images of mammary tissue
transplantation strategy is provided, with details in the Materials and Methods section. Animals were mated when they were 9–11 wk of age, and tissue
was collected on the day of parturition. B) Representative whole mounts of WT glands transplanted into AhrKO recipients exposed to vehicle (Veh) or
TCDD during pregnancy and collected on the day of parturition are shown in the top row. C) Representative whole mounts of AhrKO glands that were
transplanted into WT recipients, which were then exposed to Veh or TCDD and impregnated, are shown in the bottom row. Representative images were
obtained using a Zeiss dissecting microscope (3.13magnification) with a Nikon Coolpix E995 camera. All images are at the same magnification. D) The
relative development of transplanted glands on the day of parturition was evaluated by at least two scientists and scored on a 1–4 scale. The bar graphs
depict the average (6SEM) score of exogenous glands, and the asterisks indicate statistically significant differences between treatment groups of the same
genotype (P , 0.05, n ¼ 9). Development of transplanted mammary epithelium from WT and KO mice in vehicle-treated recipient mice did not differ
Impaired development is a consequence of both direct and indirect effects on mammary epithelium. A) A schematic representation of the MEC
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parturition (Fig. 4D). Both WT and AhrKO transplanted glands
exposed to TCDD during pregnancy had their development
impaired to some extent, with the average developmental
scores significantly lower than they were for glands collected
from animals exposed to vehicle treatment (Fig. 4, B and C).
However, compared to the consequences of AHR activation in
WT animals, the effects of TCDD on reciprocal transplants
appear less pronounced (compare the images in Fig. 1A with
Fig. 4, B and C). These observations suggest that TCDD
exposure impairs pregnancy-associated mammary development
by direct AHR-mediated effects on the epithelium and through
AHR-mediated effects in the stromal tissues, and possibly via
effects in other organs and glands as well.
Exposure to TCDD Alters the Expression of Genes
in Isolated MECs
To examine the correlation between alterations in gene
expression observed in whole tissue (Fig. 2) and epithelium,
we isolated MECs from animals exposed to TCDD on DP 0
and killed on DP 6 (Fig. 5A). In isolated MECs, there was a
300-fold increase in Cyp1a1 gene expression after exposure to
TCDD, consistent with other reports that AHR ligands induce
Cyp1a1 expression directly in mammary cells [39, 42, 43].
Interestingly, while expression of cyclin D1 was decreased in
MECs isolated from mice exposed to TCDD, the TCDD-
induced reductions in E-cadherin expression level was not
observed in isolated MECs (Fig. 5B). These results suggest that
exposure to TCDD altered the expression of cyclin D1 within
MECs, which is of interest as this is an important factor in cell
cycle progression. The lack of effect of TCDD on E-cadherin
expression levels in isolated MECs suggests that alterations in
E-cadherin after AHR activation may be related to AHR-
mediated cues from cells of the mammary fat pad or other
signals extrinsic to MECs.
The present study demonstrates that the deleterious
consequences of TCDD on pregnancy-associated mammary
development and lactogenesis occur through local effects on
the mammary gland and that AHR activation in both the
epithelial parenchyma and in the associated stromal tissue are
required for this impairment. Understanding how environmen-
tal pollutants, such as dioxins, influence pregnancy-associated
mammary gland development provides greater insight into two
important processes: pregnancy-associated mammary gland
differentiation and AHR-regulated development. Synchronized
and controlled development of mammary glands during
pregnancy is necessary for appropriate milk production.
Reasons for lactation insufficiency are not well understood;
however, decreased milk production as a consequence of
exposure to pollutants has been proposed as a contributing
factor leading to difficulties in initiating or maintaining
breastfeeding [8, 10]. Yet few studies have been undertaken
to determine precisely how environmental toxicants affect
depicted. The number on each dot plot indicates the percentage of cells in the gated region. The preparation of single cell suspensions, antibody binding,
and gating are described in detail in Materials and Methods. Bar graphs (B–D) represent average (6SEM) gene expression levels in MECs isolated from
mammary glands collected from animals exposed to TCDD or vehicle on DP 0 and killed on DP 6. Each sample is derived from tissues that were pooled
from three different animals. Lymph nodes were not included in the tissue preparation. Averages were calculated using the DDCtapproach and normalized
against GAPDH and vehicle control. Bars with asterisks indicate statistically significant differences (P , 0.05, n¼3–4/group). Original magnification33.1
TCDD-induced changes in gene expression in isolated MECs. A) The overall strategy for fluorescence-based sorting (isolation) of MECs is
TCDD AND PREGNANCY-ASSOCIATED MAMMARY DEVELOPMENT
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mammary gland differentiation during pregnancy. In addition
to helping understand how chemicals from our environment
disrupt this process, studies of mammary gland development
during pregnancy provide insight into how AHR regulates
complex developmental processes. Mammary gland develop-
ment during pregnancy follows a pattern of proliferation and
differentiation that is quite similar to events that occur in other
organs during prenatal development [4, 27]. Thus, processes
that occur in other organs during fetal development can be
studied in an adult animal simply by impregnating the female.
Herein we used several different experimental models to better
understand how AHR activation during pregnancy deregulates
mammary development and impairs lactogenesis.
In addition to responding to xenobiotics, the AHR has been
implicated as an important regulator of growth and develop-
ment; however, the specific nature of AHR’s physiological
functions remains enigmatic [13, 17]. AHR activation by
exogenous ligands disrupts proliferation and tissue differenti-
ation in many experimental systems, and AHR-null mice have
defects in hepatic growth, decreased fertility, ovarian changes,
and abnormalities with aging [18–21]. However, the role of
AHR in normal mammary gland development remains
controversial. We show here that lack of AHR did not alter
normal mammary gland development, and homozygous AHR-
deficient dams were able to nutritionally support their
offspring. This is consistent with another report wherein
AHR-null and ARNT-null mice were able to nurse their litters
normally . Collectively, these data suggest AHR and its
dimerization partner are not required for mammary gland
development and lactation. However, others have reported that
mammary glands from AHR-null mice showed impairment in
branching and morphogenesis in an ex vivo culture system
. These differences may arise because of distinctions in
experimental procedures, physiological setting (in vivo vs. in
vitro), endpoints measured, or mouse strain. For example, the
report by Hushka et al.  used AHR-null mice that were
created by one group, while in our experiments we used
AhrKO mice created in a different manner. Also, we examined
this process in vivo, whereas they used an ex vivo system for
differentiating the tissue. Despite such differences in studies
with AhrKO mice, it remains clear that AHR activation by
exogenous ligands has a profound impact on mammary gland
Mammary gland development during pregnancy is con-
trolled by many factors that are produced systemically and
locally [4–6]. Using several experimental approaches, we
endeavored to tease apart which tissues and cell types are
directly affected by TCDD. Ex vivo culture of whole organs
revealed that TCDD acts directly on mammary glands, which is
consistent with a prior report that mammary explants treated
with 2,3,7,8-tetrachlorodibenzofuran (another potent AHR
ligand) displayed decreased lobule development . Like-
wise, direct treatment of cultured mammary epithelial cells
with TCDD impairs differentiation and b-casein induction .
Collectively, these findings in mammary tissues and cells are
consistent with reports showing that AHR ligands directly
influence the differentiation of other organs [29, 44–48]. Yet,
organs are made up of different cell types, making it somewhat
difficult in many systems to delineate the specific subsets of
cell types within an organ that are responsible for AHR-
mediated changes in differentiation. It is possible to distinguish
AHR-mediated events in mammary epithelium and stromal
tissues by reciprocal transplantation, which is an approach that
has been used to delineate the contribution of receptors and
signaling molecules in other studies [49–51]. Reciprocal
transplantation of AHRþ/þand AHR?/?mammary epithelium
revealed that local factors produced by the mammary
epithelium and stromal tissues (fat pad) both contain direct
AHR targets, which are involved in disrupting pregnancy-
associated differentiation. Future experiments using transgenic
systems to conditionally eliminate AHR in specific mammary
cell types would ultimately clarify whether epithelial or stromal
AHR in the mammary gland explains the observed changes in
In addition to demonstrating that AHR has targets within
and extrinsic to the mammary epithelium, we present here the
novel finding that whatever signaling events are triggered by
TCDD exposure requires the AHR’s DNA-binding domain.
This further supports the idea that AHR regulates mammary
gland differentiation at the level of transcriptional control. One
potential target gene is cyclin D1, which showed reduced
expression in whole mammary glands and sorted MECs from
TCDD-treated mice. Given that AHR activation decreases
MEC proliferation in early pregnancy, decreased cyclin D1 is
consistent with a defect in proliferation . Of course, one
possible explanation for reduced cyclin D1 is that that there are
simply fewer MECs. However, many genes were not changed
by TCDD treatment (data not shown), and other genes, such as
Cyp1a1, were markedly elevated by TCDD exposure. There
are two additional pieces of evidence to suggest a relationship
between AHR signaling and cyclin D1. First, the work of
others has shown that activated AHR inhibits proliferation and
induces cell cycle arrest in other types of cells and tissues; thus,
observing this in mammary glands is consistent with these
studies (reviewed in [17, 52]). In particular, AHR activation by
TCDD decreased cyclin D1 in prostate cancer cells and breast
cancer cell lines [53, 54] as well as in mouse liver  and
zebrafish caudal fin regeneration models . The second
piece of evidence stems from reports that cyclin D1 is essential
for mammary gland development during pregnancy [56, 57].
Loss of cyclin D1 leads to a paucity of alveolar cells, which fail
to functionally differentiate. In fact, mammary gland develop-
ment during pregnancy is stunted in cyclin D1-deficient mice
with a phenotype that looks strikingly similar to TCDD-treated
mice. Thus, an AHR-mediated diminution in cyclin D1
provides a possible mechanism for reduced pregnancy-
associated MEC proliferation and may explain in part the
decreased number of proliferating mammary epithelial cells in
TCDD-treated pregnant mice. However, an impact on cyclin
D1 does not rule out additional affects of TCDD on growth
factors or other regulatory molecules that are locally produced
in the mammary gland.
Indeed, we also noted that mammary glands from TCDD-
treated mice had reduced expression of E-cadherin, which is a
cell-adhesion molecule directly involved in and crucial for the
development and differentiation of epithelial cells in various
tissues, including the mammary gland . Other reports have
shown that AHR activation reduces E-cadherin protein levels
in whole mammary glands, MCF-7 cells, and SCp2 cells,
suggesting a direct effect of TCDD on MEC and on this gene in
particular . However, when we isolated MECs from
TCDD-treated pregnant mice, we did not observe decreased
E-cadherin expression levels. These contradictory results may
be due to the fact that the cell lines were cultured with other
factors and/or on Matrigel, which provides an exogenous
extracellular matrix. Timing may also be a factor in sensitivity
of E-cadherin to perturbation by AHR activation. In our prior
work, we examined E-cadherin in late pregnancy (DP 17) and
in SCp2 mammary epithelial cells, which were clonally derived
from mice at midpregnancy . In contrast, in our present
study, MECs were isolated from animals exposed to TCDD in
vivo during early pregnancy (DP 0–6). Thus, E-cadherin
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expressed by MECs during early pregnancy may not be altered
by TCDD exposure, whereas AHR may alter E-cadherin
expression in later stages of pregnancy. Alternatively, AHR-
mediated changes in E-cadherin levels may be downstream of a
direct impact of AHR on other gene targets and signaling
pathways, including gene targets that are within mammary
tissue but not that are extrinsic to the epithelial compartment.
The new findings presented here suggest that impairment of
pregnancy-associated mammary gland development after
exposure to TCDD occurs due to AHR-mediated alterations
in the normal function of the mammary epithelium and
mammary fat pad (stroma). In addition to providing new
information regarding how AHR ligands alter this orchestrated
developmental process, these findings demonstrate that mam-
mary tissues are directly targeted by a common environmental
toxicant in a manner that impedes lactogenesis. When
considering public health, the implications of this are profound.
Decreased milk production as a consequence of exposure to
environmental pollutants may contribute to poor nutrition,
especially in places where breast milk is the only food available
for the neonate and where exposures to pollution remain poorly
controlled. In addition to human health, wild animal popula-
tions are exposed to dioxins and related chemicals, which may
result in decreased milk production in these species. Another
possible implication of this work is a potential connection to
breast cancer. Although not directly examined in the work
described herein, several molecules involved in normal
mammary gland development and differentiation have been
implicated in the formation of mammary tumors . This
possible relationship is further suggested by the fact that TCDD
is a known human carcinogen, and a relationship between
exposure to TCDD and related AHR-binding pollutants and
breast cancer has been reported [61–63]. Thus, in addition to
improving our understanding of how AHR signaling and
exogenous AHR-binding pollutants influence lactogenesis, the
results from the present study suggest possible involvement of
AHR ligands in the development of breast cancer.
We thank Dr. Suzanne E. Fenton (NIEHS) for generously sharing her
expertise in mammary gland physiology and development, advice on
mammary transplant surgeries, and for thoughtful discussion of our
findings. We also thank Jill Gresens for excellent technical assistance with
the real time RT-PCR and the preparation of mammary images for this
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