Transcriptional and Spatiotemporal Regulation of
Prolactin Receptor mRNA and Cooperativity With
Progesterone Receptor Function During Ductal
Branch Growth in the Mammary Gland
RUSSELL C. HOVEY,* JOSEPHINE F. TROTT, ERIKA GINSBURG, ANITA GOLDHAR, MARK M. SASAKI,
STEVEN J. FOUNTAIN, KRIPA SUNDARARAJAN, AND BARBARA K. VONDERHAAR
Molecular and Cellular Endocrinology Section, Center for Cancer Research, NCI, NIH, Bethesda, Maryland
mammary gland is stimulated by prolactin (PRL)
and progesterone (P) acting through their recep-
tors (PRLR and PR). Analysis of mammary gland
PRLR expression revealed increasing expression
of the long form (L-PRLR) and two of the three
short forms (S1- and S3-PRLR) during puberty
that became maximal late in pubescence and
early gestation, then declined during gestation.
By contrast, S2-PRLR mRNA levels remained
constant. Examination of stromal PRLR revealed
the consistent expression of L-PRLR mRNA. By
contrast, S1-PRLR was present only in the mam-
mary fat pad of neonates, whereas high neonatal
expression of S2-PRLR became undetectable
during puberty. Stromal expression of S3-PRLR
decreased to low levels during puberty and was
undetectable during lactation and involution.
Exogenous PRL stimulated DNA synthesis in
both epithelial and adjacent stromal cells in vivo.
Distribution of PRLR mRNA in mammary epithe-
lium was homogeneous before puberty and heter-
ogeneous during puberty, gestation, and early lac-
tation. A mutual role for PRLR and PR was
suggested wherein PR mRNA increased beyond 6
weeks to maximal levels during puberty and gesta-
tion then became undetectable during lactation. In
situ hybridization revealed that PR mRNA distri-
bution is homogeneous in the ductal epithelium
before 6 weeks and heterogenous during puberty
and gestation and that PRLR and PR are similarly
distributed in the ductal epithelium. Neither hor-
mone stimulated DNA synthesis in mammary
glands of ovariectomized females while their ef-
fects interacted markedly. These results demon-
strate differential PRLR transcription by epithe-
lial and stromal cells and a similar distribution of
PRLR and PR that may facilitate the interaction
Published 2001 Wiley-Liss, Inc.†
Ductal branching within the
Key words: prolactin; progesterone; receptors;
mammary gland; ovarian steroids;
epithelial-stromal; ductal branching
Of the endocrine hormones, prolactin (PRL) fulfills a
critical role during development and secretory function
of the mammary gland (Vonderhaar, 1987). The major-
ity of information concerning PRL action on the mam-
mary gland relates to the role of PRL during milk
protein synthesis whereby it activates transcription
through specific signaling cascades (Hennighausen et
al., 1997). In addition to its lactogenic effect, PRL pro-
motes significant development of the mammary gland
epithelium, particularly during gestation (Vonderhaar,
1987). The essential role for PRL during mammogen-
esis is indicated in mice lacking PRL (Vomachka et al.,
2000) or PRLR (Ormandy et al., 1997), where both
phenotypes are characterized by a mammary ductal
tree that fails to undergo typical tertiary branching
and alveolar development during gestation.
The action of PRL is mediated by the cell-surface
PRLR, a member of the class-1 cytokine receptor su-
perfamily. To date, four isoforms of the receptor have
been identified in the mouse, each varying in the size of
its cytoplasmic domain resulting from alternative 3?-
exon splicing (Davis and Linzer, 1989; Clarke and Lin-
zer, 1993; Moore and Oka, 1993) of a single gene (Or-
mandy et al., 1998). The long form of the PRLR
transduces both a lactogenic signal through the JAK-
STAT pathway (Gouilleux et al., 1994; Han et al., 1997;
Mayr et al., 1998) and a mitogenic signal through the
MAP kinase pathway (Das and Vonderhaar, 1995). Nu-
merous other signaling pathways, including Akt may
also be activated by this receptor form (Das and
Vonderhaar, 1996a,b; Schwertfeger et al., 2000). Vari-
ous short forms of the PRLR have been identified
across species (Boutin et al., 1988; Bignon et al., 1997),
where three short forms of the receptor have been
identified in the mouse (Davis and Linzer, 1989). There
is evidence that one form, S1-PRLR, can transduce a
Grant Sponsor: US Army Medical Research and Materiel Com-
mand; Grant number: DAMD 17-99-19311.
*Correspondence to: Russell C. Hovey, PhD, Building 10, Room
5B47, National Institutes of Health, 10 Center Drive, Bethesda, MD,
20892-1402. E-mail: email@example.com
Received 10 May 2001; Accepted 20 June 2001
Published online 13 August 2001; DOI 10.1002/dvdy.1179
DEVELOPMENTAL DYNAMICS 222:192–205 (2001)
Published 2001 WILEY-LISS, Inc.
work and, as such, is in the public domain in the United States of America.
†This article is a US government
mitogenic signal (Das and Vonderhaar, 1995), whereas
in the rat, the short form acts as a dominant-negative
influence on the differentiation signal transduced by
means of the L-PRLR form (Berlanga et al., 1995; Per-
rot-Applanat et al., 1997). The four PRLR isoforms
have tissue specific expression patterns (Davis and
Linzer, 1989; Buck et al., 1992; Clarke and Linzer,
1993) and are differentially regulated within the ovary
(Clarke and Linzer, 1993), liver, and kidney (Buck et
al., 1992) during gestation and lactation. In the mam-
mary gland, L-PRLR is temporally and hormonally
regulated between early gestation (Mizoguchi et al.,
1997a), late gestation, and parturition in association
with changes in its ratio with short PRLR (Mizoguchi
et al., 1997b). However, information concerning PRLR
expression within the mouse mammary gland during
nulliparous development is limited to a report by Bera
et al. (1994) localizing PRLR mRNA to the ductal epi-
thelium and demonstrating up-regulated PRLR ex-
pression during pituitary isograft-stimulated alveolar
development. More recent evidence suggests that duc-
tal branching within the mammary gland is regulated
through extra-epithelial mechanisms and/or potential
stromal PRLR, whereas epithelial PRLR facilitate com-
plete alveolar development (Brisken et al., 1999). Epi-
thelial-stromal interactions serve critical functions
during development of the mammary gland in response
to key endocrine hormones such as those derived from
the pituitary and ovaries (Fendrick et al., 1998; Hovey
et al., 1999). However, the specific function of stromal
PRLR during PRL-stimulated development has not
Accompanying the effects of PRL on mammary gland
development during puberty and gestation is the stim-
ulation of ductal branching and alveologenesis by pro-
gesterone (P). The actions of P are initiated through
progesterone receptors (PR) in the mammary epithe-
lium (Brisken et al., 1998), where acquisition of estro-
gen (E)-inducible PR in mouse mammary epithelium
occurs at around 7 weeks of age (Fendrick et al., 1998).
The essential involvement of the two PR isoforms,
PR-A and PR-B, during branching morphogenesis and
alveolar development has been demonstrated more re-
cently by the generation of transgenic (Shyamala et al.,
1998, 2000) and knockout (Lydon et al., 1995) mouse
models. Despite this understanding, information con-
cerning the interaction between P and other mammo-
genic hormones besides E (Fendrick et al., 1998) has
Given the diverse functions of PRL during mammo-
genesis, we hypothesized that expression of the various
PRLR isoforms changes both temporally and spatially
within the developing mammary gland to differentially
regulate the effects of PRL. Furthermore, we hypothe-
sized that these spatiotemporal changes are similar for
PR and that P and PRL interact to stimulate the
growth of ductal branches within the mammary gland.
Our results indicate that PRL stimulates both epithe-
lial and stromal proliferation within the mammary
gland coincident with the regulated expression of
PRLR mRNA in epithelial and stromal compartments.
Furthermore, PRLR and PR are similarly distributed
within the ductal epithelium of the mammary gland,
whereas their ligands interact to stimulate epithelial
and stromal proliferation in ovariectomized females.
Expression of PRLR mRNA Isoforms Within the
Normal Mammary Gland
By using semiquantitative RT-PCR, we examined
the gene expression profiles for the four described
mouse PRLR isoforms within the mammary gland dur-
ing its development and functional differentiation (Fig.
1). Expression of S1-PRLR was low before puberty,
increasing between 6 and 8 weeks of age to maximal
levels in the fully developed nulliparous mammary
gland at 10–12 weeks (Fig. 1A). Thereafter, mRNA
abundance declined during gestation, was low during
lactation, then sustained elevated levels during invo-
lution. By comparison, less marked changes in S2-
PRLR expression were observed during development,
where a minor increase in mRNA abundance was seen
only during the mature nulliparous state and then
increased substantially during involution (Fig. 1B).
The profile for S3-PRLR expression more closely re-
sembled that for S1-PRLR, wherein levels increased
during puberty, declined to low levels during late ges-
tation and lactation, then became elevated during in-
volution (Fig. 1C). The expression pattern of L-PRLR
mRNA was similar to that of S1-PRLR and S3-PRLR.
Abundance of L-PRLR mRNA increased between 6 and
8 weeks of age and was maximal at 10 and 12 weeks,
thereafter declining during gestation (Fig. 1D). Com-
pared with mRNA levels for S1-, S2- and S3-PRLR,
L-PRLR levels were initially increased after parturi-
tion, declined late in lactation, then increased mark-
edly during involution, consistent with previous re-
ports (Mizoguchi et al., 1997a,b). The up-regulated
expression of specific PRLR mRNAs after the onset of
puberty and during early gestation was associated with
increased expression of PRLR mRNA as confirmed by
in situ hybridization and not simply the increased pro-
portion of epithelial tissue within the developing mam-
mary gland (data not shown).
Expression of PRLR mRNA Isoforms Within the
Mammary Fat Pad
To investigate a hypothesized role for stromal PRLR,
we examined expression of PRLR mRNA in the mam-
mary stroma by using surgically prepared cleared
mammary fat pad (CFP). Abundance of all PRLR
mRNAs in the CFP was significantly less than in the
intact mammary gland, as evidenced by the increased
cycle number required for PCR amplification (see Ma-
terials and Methods section). These low levels have
thwarted attempts to date aimed at localizing stromal
PRLR mRNA by in situ hybridization at stages other
than in neonates.
PROLACTIN AND PROGESTERONE RECEPTORS IN MAMMARY GLAND
By using RT-PCR, mRNA for all isoforms of PRLR
was detected in the mammary fat pad (Fig. 2). S1-
PRLR mRNA was only detected in the mammary fat
pad on postnatal days 7 and 10, thereafter diminishing
to undetectable levels. Levels of S2-PRLR were also
maximal in the CFP of neonates and declined to unde-
tectable levels before puberty. Similarly, abundance of
S3-PRLR mRNA was greatest in the CFP from neona-
tal females and decreased during prepuberty to low
levels during puberty. Abundance remained similar
until 12 weeks of age, thereafter becoming undetect-
able during gestation, lactation, and involution. By
contrast, the level of L-PRLR mRNA was greater than
that for S1-, S2-, and S3-PRLR and remained constant
across all stages of development.
Localization of PRLR mRNA During Mammary
By using in situ hybridization and a riboprobe com-
plementary to mRNA encoding the extracellular do-
main of all PRLR isoforms, we localized PRLR expres-
sion in the developing normal mammary gland. Before
puberty, PRLR mRNA was expressed homogeneously
in epithelial cells of the mammary rudiment as well as
in adipocytes and stromal cells of the mammary fat pad
(Fig. 3A). During early puberty (at 6 weeks of age), the
expression of PRLR mRNA was homogeneous in the
mammary epithelium, whereas expression in the
stroma became undetectable (Fig. 3B). Within the
mammary glands of pubertal females, PRLR mRNA
localized specifically to the body cells, but not cap cells,
of the ductal terminal end bud (Fig. 3C). After the onset
of puberty between approximately 6 and 8 weeks of
age, expression of PRLR mRNA was up-regulated and
became heterogeneously distributed within the ductal
epithelium (Fig. 3D). This distribution of expression
was also evident during early gestation (Fig. 3E). To-
ward the end of gestation (between days 18 and 21), the
distribution of PRLR mRNA became more homoge-
neous within the developing alveolar epithelium,
murine mammary gland during postnatal mammary development. Mam-
mary tissue was pooled from at least eight female mice killed at the
indicated stages. Total RNA was reverse transcribed then amplified by
polymerase chain reaction (PCR) by using primers specific for the
various PRLR forms. A: S1-PRLR, short form 1; B: S2-PRLR, short
Profile of prolactin receptor (PRLR) gene expression within theform 2; C: S3-PRLR, short form 3; D: L-PRLR, long form. Quantified
values were expressed relative to the corresponding abundance of glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Values are
representative of at least two reverse transcription reactions that were
amplified at least twice by PCR. D, diestrus; E, estrus.
HOVEY ET AL.
whereas a subpopulation of cells continued to express
increased levels of PRLR mRNA. By contrast, the dis-
tribution of PRLR mRNA remained heterogeneous
within the ductal epithelium at this stage (Fig. 3F).
This pattern of distribution was still evident on day 1 of
lactation (Fig. 3G), whereas by day 10 of lactation the
expression of PRLR mRNA was homogeneous in the
ductal and alveolar epithelium (Fig. 3H). The hetero-
geneous distribution of PRLR mRNA within the mam-
mary gland on day 1 of lactation was shown to corre-
spond to a similar distribution of L-PRLR mRNA by
using an isoform-specific riboprobe (Fig. 4). Efforts to
localize the expression of S1-, S2-, and S3-PRLR mRNA
so far have been hindered by sensitivity limitations.
Stimulation of Epithelial and Stromal
Proliferation by Exogenous PRL
To identify specific cell targets for PRL-induced pro-
liferation, we performed [3H]TdR autoradiography of
mammary tissue from 4- and 10-week-old nulliparous
female mice treated with oPRL for 5 days. Exogenous
oPRL stimulated a 2.6-fold and 5.2-fold increase in
epithelial proliferation relative to controls at 4 and 10
weeks, respectively (Fig. 5A; P ? 0.05). We also exam-
ined whether exogenous PRL stimulated stromal pro-
liferation within the mammary gland. After PRL treat-
ment, labeling of stromal cells within 50 ?m of the
ductal epithelium in ovary-intact females was in-
creased 2.6-fold and 5-fold in 4- and 10-week-old fe-
males, respectively (Fig. 5B; P ? 0.05). This increase
reflected increased DNA synthesis in both adipocytes
and fibroblasts. These results indicate that PRL in-
duces the proliferation of both epithelial and stromal
cells within the mammary glands of prepubertal and
peripubertal intact mice.
Duration of PRL Exposure Alters Proliferative
Responses Within the Mammary Gland
Having demonstrated PRL-induced epithelial and
stromal proliferation in the mammary glands of nullip-
arous female mice, we established the temporal prolif-
erative response to oPRL in ovary-intact mice (Fig. 6).
Mice treated with excipient alone for 1 to 12 days
demonstrated a consistent baseline level of epithelial
and stromal proliferation. By comparison, PRL in-
creased epithelial proliferation after exposure for 1 to 5
days, whereas treatment for 12 days failed to sustain
this epithelial proliferation and resulted in a return to
basal levels (P ? 0.05). Stromal proliferation was in-
duced only after 3 and 5 days of treatment and, similar
to the epithelial response, was not sustained to 12
Developmental Expression and Distribution of
mRNA for PR Is Similar to That for PRLR
Given our hypothesis that PRLR and PR expression
and function are coordinated, the profile of total PR
mRNA expression during development of the mam-
mary gland was examined (Fig. 7). Analysis by RT-PCR
demonstrated stage-specific regulation of PR mRNA,
epithelium-free mammary fat pad during postnatal mammary develop-
ment. Epithelium-free mammary fat pads surgically prepared at 3 weeks
of age were pooled from at least six female mice at the indicated stages.
Tissue collected before 3 weeks of age was directly collected from the
epithelium-free portion of the mammary gland. Total RNA was reverse
transcribed then amplified by polymerase chain reaction (PCR) using
Expression of prolactin receptor (PRLR) mRNAs within theprimers specific for the indicated PRLR short forms (S1-, S2-, and S3-
PRLR) and the long form (L-PRLR), electrophoresed, then stained with
ethidium bromide. Equivalence of RNA loading was assessed by PCR
amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Values are representative of two reverse transcription reactions amplified
by PCR. preg., pregnant; lact., lactating; invol., involuted; D, diestrus; E,
PROLACTIN AND PROGESTERONE RECEPTORS IN MAMMARY GLAND
where levels were undetectable before 3 weeks of age
then increased markedly after 6 weeks of age to reach
maximum levels in the mammary glands of mature
nulliparous and early pregnant females. Thereafter,
levels declined during gestation to become undetect-
able during lactation then subsequently increased dur-
Localization of PR mRNA by in situ hybridization
confirmed this profile of gene expression. In prepu-
bescent females before approximately 6 weeks of age,
mRNA within the mammary gland localized by in situ hybridization. A
35S-labeled antisense riboprobe corresponding to the common extracel-
lular domain of the mPRLR was hybridized with sections of mammary
tissue from the following developmental stages: A: 3 weeks; B: 6 weeks;
Spatiotemporal distribution of prolactin receptor (PRLR)
C: terminal end bud, 6 weeks; D: 12 weeks; E: day 3 gestation; F: day 18
gestation; G: day 1 lactation; H: day 10 lactation. Sections hybridized with
a sense strand probe indicated the absence of nonspecific signal (refer
Fig. 8I). Scale bar in H ? 100 ?m in A,B,D–H; 50 ?m in C.
HOVEY ET AL.
the distribution of PR mRNA was homogeneous
within the ductal epithelium. Specific analysis of
gene expression within terminal end buds identified
that PR mRNA, similar to PRLR mRNA, localized to
body cells but not cap cells (compare Fig. 8A,B).
Thereafter, the distribution of PR mRNA within the
ductal epithelium became heterogeneous and punc-
tate in mature nulliparous females in association
with increased mRNA expression by individual cells
(Fig. 8D). This heterogenous distribution was also
evident in the ductal epithelium during early gesta-
tion (Fig. 8F). In late gestation, the majority of alve-
olar epithelial cells showed a low level of homoge-
neous PR expression with occasional ductal and
alveolar cells having increased levels of PR mRNA
(Fig. 8H). By contrast, PR mRNA was undetectable
within the lactating mammary gland (Fig. 8I). To
determine the potential coassociation between PR
and PRLR, in situ hybridization for these receptors
was performed on serial sections of mammary tissue.
Further to the demonstration of similar mRNA dis-
tribution in the terminal end bud (Fig. 8A,B), anal-
ysis of mammary glands from mature nulliparous
and pregnant females showed that both receptors are
coassociated in the mammary ductal epithelium
(compare Figs. 8C,E,G with Figs. 8D,F,H, respec-
Synergistic Stimulation of Epithelial
Proliferation by P and PRL
The coassociation between PRLR and PR in the duc-
tal epithelium and the ability of PRL and P to stimu-
late epithelial proliferation in the mammary gland of
intact females (current results and Atwood et al., 2000)
prompted us to test whether these hormones synergize
to stimulate epithelial and stromal proliferation alone
or in the presence of E. Ovariectomized mice at 10
weeks of age displayed a low basal level of epithelial
proliferation relative to intact females (Fig. 9; 0.07%
compared with 0.7%, Fig. 5A). Treatment with E in-
creased epithelial proliferation 130-fold (P ? 0.05),
whereas P and PRL were without effect (P ? 0.05).
Combined treatment with E?P did not alter epithelial
proliferation relative to E alone, whereas PRL sup-
pressed E-induced proliferation by 60% (P ? 0.05).
Although P and PRL alone were without effect, they
markedly interacted (P ? 0.001) to increase prolifera-
tion, primarily in the ductal epithelium, by almost 400-
fold above that in saline-treated females. This response
was reduced by approximately 50% (P ? 0.05) when E
was also administered. Proliferation indices also re-
vealed hormonal effects on stromal proliferation. Inter-
estingly, basal stromal proliferation was similar in
ovariectomized and intact females (0.53% compared
mRNA within the mouse mammary gland. Tissue sections from female
mice at (A) 12 weeks; (B) day 1 lactation, and (C) day 10 lactation were
hybridized with a
mRNA encoding the intracellular domain of L-PRLR. Arrows in B indicate
Distribution of prolactin receptor (PRLR) long form (L-PRLR)
35S-labeled antisense riboprobe complementary to
the increased expression of L-PRLR mRNA in specific epithelial cells.
D: Corresponding section for C hybridized with the sense control ribo-
probe showing the absence of nonspecific hybridization. Scale bar ? 100
?m in D (applies to A–D).
PROLACTIN AND PROGESTERONE RECEPTORS IN MAMMARY GLAND
with 0.67%, see Fig. 5B). Estrogen induced an eightfold
increase (P ? 0.05) in stromal proliferation, whereas P
and PRL were without effect (P ? 0.05). Combined
treatment with E?P failed to increase stromal prolif-
eration relative to E alone (P ? 0.05), whereas PRL
suppressed E-induced proliferation by 45% (P ? 0.05).
Similar to the response observed in epithelial cells, P
and PRL synergized (P ? 0.001) to increase stromal cell
division by 17-fold relative to saline controls. In con-
trast, stromal proliferation induced by P?PRL was not
as sensitive to the negative effect of E recorded in
PRL functions as a major regulator of mammary
gland growth and function (Vonderhaar, 1987). Al-
though these effects are undisputed, PRLR expression
and distribution within the mammary gland, particu-
larly during development in nulliparous females, has
not been widely examined. Furthermore, although a
role for PRLR within the mammary fat pad has been
suggested (Brisken et al., 1999), PRLR expression
within the mammary stroma has not been reported.
The present results indicate a significant role for
PRL and PRLR during the growth of ductal branches in
the nulliparous female mouse. Specifically, short-term
PRL treatment of ovary-intact females stimulated ep-
ithelial proliferation within the ductal epithelium of
both prepubertal and pubescent females as determined
PRL and PRLR leads to impaired ductal branching and
failed alveolar development (Ormandy et al., 1997; Vo-
machka et al., 2000). This concurs with the demonstra-
tion that PRL is mitogenic for primary mouse mam-
mary epithelial cells in vitro (Edery et al., 1984). In the
present study, epithelial and stromal proliferation in
response to exogenous PRL depended upon the period
3H-TdR autoradiography. Conversely, ablation of
and (B) stromal cell proliferation within the mammary glands of nullipa-
rous BALB/c mice at 4 and 10 weeks of age. Mice administered twice-
daily injections of saline (SAL) or oPRL (1 ?g/g BW) for 5 days received
100 ?Ci [3H]thymidine 2 hr before being killed. Autoradiography was
performed on paraffin sections and the percentage of labeled cells de-
termined. Data are means ? SEM (n ? 6-7/group). *P ? 0.05 vs.
respective SAL-treated mean.
Effect of exogenous prolactin receptor (oPRL) on (A) epithelial
liferation within the mammary glands of nulliparous pubescent mice.
Females at 10 weeks of age were administered oPRL (1 ?g/g body
weight) or saline (SAL) twice-daily for the indicated periods and received
100 ?Ci [3H]thymidine 2 hr before killing. Autoradiography was performed
on paraffin sections, and the percentage of labeled epithelial (A) and
stromal (B) cells were determined. Data are means ? SEM (n ?
6/group). *P ? 0.15, **P ? 0.05 vs. respective SAL mean.
Time course of prolactin receptor (PRL)-induced cellular pro-
HOVEY ET AL.
of PRL exposure. Stimulation of epithelial proliferation
within 24 hr of PRL exposure indicates that PRL ini-
tiates a rapid mitogenic response, whereas intrigu-
ingly, prolonged exposure for 12 days failed to sustain
increased proliferation. This latter response contrasts
to demonstrations by others that chronic PRL exposure
stimulates alveolar development (Nagasawa et al.,
1985). Such refractoriness to prolonged PRL treatment
may reflect the length of treatment or the dose admin-
istered, consistent with the demonstration that other
factors such as estrogen (Nagasawa and Yanai, 1971)
are essential for sustained PRL-induced proliferation.
Alternatively, it is tenable that ovine PRL differs in its
ability to induce prolonged proliferation relative to
mouse PRL, perhaps due to the development of a
chronic immune response against exogenous oPRL.
Given that mammary glands of PRL- and PRLR-
deficient mice develop a ductal tree, it is unlikely that
PRL is critical for ductal elongation per se. However,
our results indicate that PRL can stimulate the prolif-
eration of mammary ductal epithelium at 4 weeks of
age during a phase of allometric ductal elongation. It is
possible that the contribution of PRL to normal ductal
elongation is minimal and can be offset by other hor-
mones that are essential for ductal development such
as E and growth hormone (GH; Kleinberg, 1997). In
addition, ductal elongation represents an event distinct
from ductal branching (Atwood et al., 2000). Based on
the results of gene ablation studies, the present results
support the concept that PRL, similar to P, may spe-
cifically regulate ductal branching rather than elonga-
tion within the mammary gland (Atwood et al., 2000).
Although a previous report localized PRLR mRNA to
the ductal epithelium (Bera et al., 1994), the resolution
of those results restricted conclusions to the finding
that PRLR mRNA expression is up-regulated during
PRL-induced alveolar budding. Our present results ex-
tend this observation to reveal that the ductal epithe-
lium assumes a heterogeneous distribution of PRLR
between 6 and 8 weeks of age that is maintained into
gestation. This change in PRLR distribution coincides
with increased ovarian steroid-responsiveness (Fen-
drick et al., 1998) and the initiation of tertiary ductal
branching and alveolar budding. What remains to be
determined is the precise contribution of the various
PRLR forms to this process. Recently, we have shown
that the distribution of PR-positive cells also becomes
heterogeneous during this period (Seagroves et al.,
2000) as a likely response to E (Fendrick et al., 1998)
mRNA within the mouse mammary gland and coassociation with prolac-
tin receptor (PRLR) mRNA. Serial sections of mammary tissue were
hybridized with a
mRNA encoding all PRLR forms (A,C,E,G) or both forms of PR
(B,D,F,H,I). Mammary tissues were from female mice at either 6 weeks
(A,B), 12 weeks (C,D), day 3 gestation (E,F), day 18 gestation (G,H), or
day 10 lactation (I). J: The absence of nonspecific hybridization with the
sense strand PR probe. Scale bar ? 100 ?m in J (applies to A–J).
Spatiotemporal expression of progesterone receptor (PR)
35S-labeled antisense riboprobe complementary to
Fig. 7. Profile of progesterone (PR) gene expression within the intact
mammary gland during postnatal development. Mammary tissue sam-
ples prepared as described in Fig. 1 were subjected to polymerase chain
reaction (PCR) analysis by using primers that amplify mRNA encoding
both isoforms of PR. Quantified intensities were expressed relative to the
amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
amplified by reverse transcriptase-PCR. Values are representative of at
least two reverse transcription reactions amplified by PCR. Preg., preg-
nant; lact., lactating; invol., involuted; D, diestrus; E, estrus.
PROLACTIN AND PROGESTERONE RECEPTORS IN MAMMARY GLAND
and may regulate branching morphogenesis (Atwood et
al., 2000). The present demonstration that PRLR and
PR are coassociated in the ductal epithelium extends
this proposal to suggest that the distribution of both
PR and PRLR dictates branching growth in the mam-
mary gland. This cooperativity is firmly emphasized by
the synergistic effects of P and PRL on epithelial pro-
liferation in vivo. A similar interactive response has
been demonstrated in doubly operated rats (Stou-
demire et al., 1975) consistent with the demonstration
that P and PRL synergize to stimulate mammary epi-
thelial cell proliferation in vitro (Imagawa et al., 1985).
That this response was accompanied by increased pro-
liferation in stromal cells suggests a role for epithelial-
stromal interactions in this response. It is well estab-
lished that P, similar to PRL, is requisite for alveolar
development (Lydon et al., 1995), where overexpres-
sion of PR results in precocious branching morphogen-
esis and alveolar development (Shyamala et al., 1998,
2000). Similarly, exogenous P stimulates epithelial
proliferation in peripubertal (Atwood et al., 2000) and
pubertal (Haslam, 1988) female mice. Previous reports
have demonstrated that PR primarily localize to the
mammary epithelium (Shyamala et al., 1997; Sea-
groves et al., 2000), despite the disputed presence of
stromal PR (Humphreys et al., 1997; Fendrick et al.,
1998) that likely exist in the capsular connective tissue
of the mammary fat pad (Shyamala et al., 1997). Re-
cently, we have shown that PR-positive epithelial cells
are nonproliferative in the nulliparous mammary
gland (Seagroves et al., 2000), consistent with demon-
strations that dividing cells do not simultaneously ex-
press ER or PR (Clarke et al., 1997; Saji et al., 2000). In
keeping with a mutual role for PR and PRLR, mice
lacking c/EBP-? overexpress PR (Seagroves et al.,
2000) coincidentwith overexpression
whereas mice lacking PR have reduced PRLR expres-
sion (Hovey et al., unpublished data). Despite these
results, analysis of PRLR in PR-A transgenic mice
failed to demonstrate any effect of overexpressed PR-A
on PRLR levels (Shyamala et al., 1999), whereas PR
knockout mice maintain PRL responsiveness with re-
spect to milk protein expression (Lydon et al., 1999).
Results describing a negative effect of P on PRLR ex-
pression (Djiane and Durand, 1977) need to be care-
fully evaluated in the present context, because the late
pregnant mammary gland undergoes a transition from
being P-sensitive to P-insensitive during the onset of
lactogenesis when PRLR activity and function also
changes. This change coincides with the finding of our-
selves and others (Haslam and Shyamala, 1980) that
PR mRNA is down-regulated at parturition while
PRLR mRNA levels are maintained. Previously, it has
been proposed that PRL stimulates mammary gland
proliferation indirectly by increasing luteal secretion of
P (Bocchinfuso et al., 2000). The present studies in
ovariectomized females clearly indicate that P and PRL
converge at the level of the mammary gland to stimu-
late epithelial proliferation independent of ovarian
function. However, the hierarchy of P and PRL signal-
ing in the mammary epithelium during branching mor-
phogenesis remains unclear. Results from in vitro stud-
ies indicate cross-talk between PR and PRLR signaling
pathways, where breast cancer cells regulate PR ex-
pression through MAP-kinase (Lange et al., 2000) that
functions downstream of the PRLR.
The present results indicate a role for stromal PRLR
during PRL-induced epithelial proliferation. Although
an early report from this laboratory indicated that
125I-PRL did not bind to microsomes from the cleared
mammary fat pad (Bhattacharya and Vonderhaar,
1979), this finding may reflect the age of the females or
the sensitivity of the assay. Conversely, recent evi-
dence from heterologous transplantation experiments
provides compelling evidence for functional stromal
(Brisken et al., 1999). Consistent with this proposal, we
demonstrate that PRL-induced epithelial proliferation
occurs before PRL-induced stromal cell division. This
contrasts to the action of E whereby the proliferation of
mammary fibroblasts precedes that of epithelial cells,
suggestive of paracrine stimulation from fibroblasts
(Shyamala and Ferenczy, 1984). The modulatory sig-
nals between epithelial and stromal cells during PRL-
stimulated proliferation remain undefined. Our find-
epithelial cells initiate proliferation by stromal cells, or
alternatively, induce their responsiveness to PRL. As-
sociated with the demonstration of stromal PRLR is
tin (PRL) on cellular proliferation within the mammary glands of ovariec-
tomized nulliparous mice. Females ovariectomized at 9 weeks of age
were treated for 5 days with combinations of oPRL (1 ?g/g body weight),
17?-estradiol (E; 1 ?g) and progesterone (P; 1 mg) twice per day in saline
(SAL) excipient. Mice received 100 ?Ci [3H]thymidine 2 hr before killing,
and the percentage of labeled epithelial (open bars) and stromal cells
(filled bars) was determined from autoradiograms. Data are means ?
SEM (n ? 6-7/group). a–d: Means with different superscripts for a given
cell type (epithelium or stroma) are significantly different, P ? 0.05.
Effect of various combinations of ovarian steroids and prolac-
PROLACTIN AND PROGESTERONE RECEPTORS IN MAMMARY GLAND
the stage-specific regulation of PRLR isoform expres-
sion within the mammary fat pad. Interestingly, L-
PRLR was consistently expressed throughout develop-
ment, whereas the S-PRLR forms declined at various
rates in nulliparous females. To date, roles for the
various S-PRLR forms have not been clearly defined.
Fibroblasts transfected with S1-PRLR proliferate in
response to PRL (Das and Vonderhaar, 1995), whereas
the rat S-PRLR that is homologous to the murine S3-
PRLR, functions as a dominant-negative receptor dur-
ing PRL-induced differentiation (Perrot-Applanat et
al., 1997). By contrast, L-PRLR may function in a more
general sense as a regulator of adipocyte function given
that PRL is involved in adipocyte differentiation
(McAveney et al., 1996) and the L-PRLR induces fibro-
blasts to accumulate lipid in vitro (Nanbu-Wakao et al.,
2000). A recent report confirms that mRNA encoding
various forms of the PRLR are present in adipose tis-
sue and are regulated during various developmental
states (Ling et al., 2000).
Taken together, these results indicate a role for PRL
acting through stromal and epithelial PRLR during the
growth of ductal branches in the mammary gland. The
associated distribution of PRLR and PR and their co-
ordinated expression facilitates a synergistic effect of
PRL and P to regulate branching growth and alveolo-
genesis in a specific population of epithelial cells.
Animals, Treatments, and Tissues
All procedures were conducted in accordance with
the NIH Guide for the Care and Use of Laboratory
Animals. BALB/c mice were maintained on a 12 hr
light/12 hr dark schedule with ad libitum access to
laboratory chow and water.
Mice were killed during puberty at estrus and
diestrus as determined by vaginal appearance (Cham-
plin et al., 1973) and confirmed by vaginal lavage
(Rugh, 1967) to address possible hormone-induced
variations. Mice killed during gestation were mated at
10 weeks of age, and the presence of a vaginal plug was
considered day 0 of gestation. Litter size was standard-
ized to six to eight pups. Weaning was performed on
day 10 of lactation and was referred to as day 0 of
involution. Lymph nodes were removed from all intact
mammary tissue at harvest.
To study changes in the expression of PRLR mRNA
within the mammary fat pad during development, en-
dogenous mammary epithelium was surgically re-
moved to leave a CFP (Faulkin and DeOme, 1960).
Briefly, female mice at 3 weeks of age were anesthe-
tized with avertin (2, 2, 2-tribromoethanol, Aldrich
Chemical Company, Milwaukee, WI), and the abdomi-
nal #4 mammary glands were exposed through a ven-
tral Y-shaped incision. The lymph node and nipple-
associated epithelial tissue was removed from the #4
mammary gland by cautery, leaving a CFP. Mice were
recovered and subsequently killed at various stages of
development. Mice killed in lactation had nursed six to
eight pups from the unoperated mammary glands. Ep-
ithelium-free mammary fat pads were obtained from
unoperated females at 3 weeks of age and less by col-
lecting the region of mammary fat pad dorsal to the
supramammary lymph node. All tissues were snap-
frozen in liquid nitrogen and stored at ?80°C.
Nulliparous BALB/c mice were treated with PRL to
examine its effect on mammary gland cell proliferation.
Females at 4 or 10 weeks of age were injected twice a
day with oPRL (1 ?g/g body weight [BW]) or saline
excipient in 0.1 ml volume (s.c.) for various intervals
between 1 and 12 days. In a separate experiment, mice
were bilaterally ovariectomized at 9 weeks of age under
avertin anesthesia. Mice were treated with various
hormone combinations 1 week later when circulating
ovarian steroid levels had declined, whereas the mam-
mary gland remained sensitive to hormonal stimula-
tion (Haslam, 1988). Treatments were various combi-
nations of 17?-estradiol (1 ?g), P (1 mg), and oPRL (1
?g/g BW) administered twice daily for 5 days as s.c.
injections in saline excipient (0.25 ml).
Autoradiography and Labeling Index
Two hours before killing, mice used for cell prolifer-
ation experiments were injected i.p. with 100 ?Ci
[methyl-3H]thymidine (3H-TdR; 25 Ci/mmol, Amer-
sham Pharmacia, Arlington Heights, IL) in a final vol-
ume of 0.1 ml. Tissue was fixed by using 4% parafor-
sections (4 ?m) of mammary glands from radiolabeled
mice were dewaxed, dehydrated, and dipped in NTB-2
emulsion (Eastman Kodak, Rochester, NY). Tissues
were exposed to emulsion for 4 days at 4°C then devel-
oped and fixed. Slides were counterstained with nu-
clear fast red (Vector, Burlingame, CA) and cover-
Epithelial and stromal cell proliferation was deter-
mined by counting at least 1,500 epithelial or stromal
cells stained with nuclear fast red at 400? magnifica-
tion. Labeling index was described as the percentage of
cells that were covered by at least five silver grains.
Stromal cells evaluated for [3H]TdR incorporation were
classified as those cells (fibroblasts and adipocytes)
within 50 ?m of the ductal epithelium.
Samples of intact and epithelium-free mammary
gland tissue pooled from at least eight mice per devel-
opmental stage were homogenized in Trizol (Life Tech-
nologies, Gaithersburg, MD) and total RNA extracted.
Total RNA was then treated with DNAse I (Life Tech-
nologies) before phenol:chloroform purification to re-
move any contaminating genomic DNA.
HOVEY ET AL.
A semiquantitative approach was used to evaluate
changes in receptor gene expression within the devel-
oping mammary gland. We have performed extensive
validation of this method previously and have shown
that it yields results comparable to those obtained by
competitive RT-PCR. All primer pairs span intronic
sequence and, combined with DNAse treatment of
RNA, did not amplify any products from genomic DNA.
Each experiment included appropriate negative con-
trols that confirmed the absence of contamination dur-
ing PCR. For each primer pair, we determined the
range of PCR cycles that facilitated linear amplifica-
tion in all samples analyzed, and subsequently per-
formed amplification by using the number of cycles
that afforded amplification in the linear range for all
DNAse-treated RNA (1 ?g) was reverse transcribed
by using MMLV reverse transcriptase (Life Technolo-
gies) primed with oligo dT and random hexamers in a
final volume of 25 ?l. First-strand cDNA (2–5 ?l) was
subjected to PCR amplification by using 0.2 ?M each
primer and PCR master mix (Roche Molecular Bio-
chemicals, Indianapolis, IN). Primer sequences specific
for the four forms of the mouse prolactin receptor were
those described by Clarke and Linzer (1993) and used a
common 5? primer, 5? AAA GTA TCT TGT CCA GAC
TCG CTG 3?, corresponding to nucleotides 585-608 of
the mPRLR gene. The number of PCR cycles was opti-
mized for each primer set to achieve amplification
within the linear range as a semiquantitative measure
of mRNA abundance. Amplification conditions, gene
specific 3? primers, and expected product sizes were as
follows, each with a final extension step of 72° for 10
min: (1) L-PRLR: 5? AGC AGT TCT TCA GAC TTG
CCC TT 3?, 279-bp product; 94°C for 30 sec; 64°C for 1
min, 72°C for 1.5 min, and intact glands, 27 cycles; CFP
40 cycles. (2) S1-PRLR: 5? AAC TGG AGA ATA GAA
CAC CAG AG 3?, 279-bp product; 94°C for 2 min; 65°C
for 2 min, 72°C for 3 min, and intact glands, 33 cycles;
CFP 40 cycles. S2-PRLR: 5? TCA AGT TGC TCT TTG
TTG TGA AC 3?, 279-bp product; 94°C for 30 sec; 64°C
for 1 min, 72°C for 1.5 min, and intact glands, 34 cycles;
CFP 40 cycles. (3) S3-PRLR: 5? TTG TAT TTG CTT
GGA GAG CCA GT 3?, 279-bp product; 94°C for 30 sec;
64°C for 1 min, 72°C for 1.5 min, and intact glands, 29
cycles; CFP 40 cycles.
Samples of pooled RNA were analyzed by semiquan-
titative RT-PCR, allowing us to assay and directly com-
pare multiple stages of mammary development within
a single experiment. Previously, we have determined
between-animal variation for the expression of the dif-
ferent PRLR isoforms in mammary tissue at specific
states during nulliparous, pregnant, and lactational
development. For five individual animals at these
stages, it was determined that the SEM, as a percent-
age of the mean, was ?13% for PRLR-L mRNA, ?23%
for S1-PRLR, and ?33% for S2- and S3-PRLR mRNA.
Expression of PR mRNA was determined by using
primers spanning the mPR gene that amplify both A
and B isoforms between nucleotides 2389 and 2783 to
give a 395-bp product: PR 5?: 5? CCC ACA GGA GTT
TGT CAA ACT C 3?; PR 3?: 5? GTC ATC ACT TTT TGT
GAA AGA GGA GCG GC 3?; 94°C for 1 min; 55°C for 2
min, 72°C for 2 min, and intact glands, 29 cycles.
PCR products were resolved by agarose gel electro-
phoresis, stained with ethidium bromide, and their
intensity quantified by using NIH Image. Gene expres-
sion levels were expressed relative to the respective
level of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA determined after 22 cycles by using
primers and conditions obtained from Clontech (Palo
Alto, CA). GAPDH was selected as the housekeeping
gene for normalization given our observations that it
remains relatively constant, as a function of total RNA,
during mammary gland development. Gene expression
levels were determined from PCR reactions performed
on each of at least two RT reactions.
In Situ Hybridization
A riboprobe complementary to all PRLR forms was
generated from a 318-bp PRLR cDNA (corresponding
to the extracellular region common to all PRLR forms)
subcloned into PCRScript (Stratagene, Cedar Creek,
TX). The L-PRLR form was localized by using a ribo-
probe complementary to mRNA encoding its cytoplas-
mic domain. Templates were linearized with either
EcoRV or NotI for the synthesis of cRNA by using T3 or
T7 polymerase in the presence of35S-UTP (Amersham
Pharmacia, Arlington Heights, IL). In situ hybridiza-
tion was performed as described (Seagroves et al.,
2000). Briefly, paraffin sections (4 ?m) were mounted
on silanized slides (ProbeOn, Fisher Scientific, Pitts-
burgh, PA), dewaxed, and pretreated with 15 ?g/ml
proteinase K and acetic anhydride (0.25% in 0.1 M
triethanolamine). Sections were hybridized with cRNA
probes (1 ? 105cpm/?l) then washed to 0.1 ? SSC and
treated with 20 ?g/ml RNAseA. Slides were dipped in
emulsion (NTB-2, Kodak, Rochester, NY), exposed for
6–8 weeks, and counterstained with nuclear fast red.
Statistical analyses were performed by using proce-
dures within StatView. Means comparisons were per-
formed by analysis of variance and Fisher’s least sig-
nificant difference test or by one-sided Student’s t-test.
R.C.H. is supported by the U.S. Army Medical Re-
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