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Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Estrogen (E) and progesterone (P), in cooperation with pituitary hormones, are the primary systemic hormones required for the induction of proliferation and differentiation of epithelial and stromal cells leading ultimately to the formation of ductal and alveolar structures during mammary gland development. The interaction of E and P with GH, PRL, and insulin in regulating this differentiative process has been well documented (1). Steroid hormones also regulate the expression of a number of different locally acting growth factors, including members of the epidermal growth factor, insulin-like growth factor, and fibroblast growth factor (FGF) families (2, 3). Most of these growth factors exhibit localized effects due to protein stability, adhesion and residence in extracellular matrix, transport and secretion, and availability of receptor molecules. For this reason they are believed to act as local mediators of the differentiative and proliferative signals of the systemic hormones. Systemic regulation of locally acting growth factor activity allows for fine regulation of large-scale developmentally associated proliferative and differentiative functions.
Mammary gland development is dependent on physical, molecular, and often reciprocal, interactions between the stromal and epithelial compartments (4). The ability to recapitulate fully differentiated structures from a fragment of syngeneic parenchyma, and to separate and recombine epithelial and stromal compartments in vivo, makes the mammary gland an excellent model system in which to study these interactions. Evidence for this reciprocal dependence has been demonstrated in classic recombination experiments between the epithelial and stromal androgen receptor pathways (4). The specific role of the epithelial and stromal PR in the development and differentiation of the mammary gland is unclear (5, 6).
Wnt-1, the progenitor of a family of related growth factors, was discovered in mouse mammary tumors as a result of proviral activation (7). Members of the Wnt gene family are expressed in invertebrates and vertebrates where they regulate cell fate and pattern formation (8). Wnt genes, other than Wnt-1, are expressed in the mammary glands of mice in a developmentally specific pattern (9, 10, 11). The function of these endogenous Wnt genes during mammary gland development is unknown. From these studies it is apparent that Wnt gene expression is tightly regulated and is dependent on the developmental state of the mammary gland. In BALB/c mice, Wnt-2 is expressed primarily during early ductal development, 5–8 weeks postnatally, coincident with time of PR induction by E, and is markedly down-regulated at the onset of pregnancy. Conversely, Wnt-5B transcripts are detectable in the late virgin gland at 6–12 weeks of age but increase markedly during pregnancy, reaching a peak at day 18. Wnt-5B expression is localized primarily in the ductal and lobuloalveolar cells, while Wnt-2 expression is detected in the stroma (9, 11). These results suggest that E and P may play a role in regulating Wnt-2 and Wnt-5B gene expression in both the stroma and epithelium. This restricted pattern of gene expression is indicative of molecules that may be involved in the developmental processes of the gland.
In this study the progesterone receptor knockout (PRKO) mouse (12) has been used for reciprocal transplantation experiments in syngeneic mice to investigate the distinct roles of the stromal and epithelial PR in mammary ductal and alveolar development. Wnt-2 and -5B provided specific molecular markers of steroid hormone action in the mammary gland stroma and epithelium, respectively. The PRKO mouse permitted definition of the unique effects of P distinct from those mediated by E on Wnt gene expression. This experimental approach should facilitate the identification of other steroid-mediated local growth factors on mammary gland development.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Interestingly, the PR-/-
epithelium failed to develop alveoli and to display an increase in the
number of secondary ductal branches in response to steroid hormone
treatment (Fig. 1B). The control ipsilateral glands from the host
animal responded as expected to steroid hormone treatment with alveolar
proliferation (Fig. 1, D vs. C). This result demonstrates
that the PR in the epithelium is required for normal lobuloalveolar
formation and differentiation of the epithelium. In addition, the
presence of PR-regulated signaling pathway in the stroma cannot
compensate for the lack of PR in the epithelium. In contrast,
PR+/+ epithelium transplanted into the cleared fat pad of
PR-/- hosts and treated with E and P
exhibited lobuloalveolar development (Fig. 2D). An
increase in secondary branching in these E- and P-treated transplants
can be clearly seen under higher magnification (Fig. 2F, arrow). However, an unexpected, marked reduction in the
extent of ductal outgrowth was observed in these transplants after 10
weeks of growth (Fig. 2C), as compared with the PR+/+ (Fig. 2, A and B) and the PR-/- (Fig. 1A)
epithelium transplanted into the PR+/+ stroma. The same
PR+/+ epithelium transplanted in PR+/+ stroma
responded as expected to steroid hormone treatment with extensive
alveolar growth and an increase in secondary branching (Fig. 2B). The
outgrowths from the PR+/+ epithelium transplanted into
the PR-/- stroma also displayed unusual
terminal endbuds (Fig. 2, C and E).
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). A 2-fold decrease (P
< 0.002) in Wnt-2 gene expression is observed after only 2 days of E
and P treatment. Wnt-2 gene expression decreased progressively with
daily E and P treatment and remained low to day 12 (data not shown).
This decrease in gene expression of Wnt-2 is not, however, as dramatic
(20-fold) as that observed after the onset of pregnancy (9).
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Interestingly, mice treated with E alone showed the same 5-fold
decrease relative to day 1 E alone mice in Wnt-2 gene expression after
14 days (n = 3, P < 0.001).
Wnt-5B Expression Is Induced by E and P in ovx and Intact Mice
During normal mammary gland development, Wnt-5B expression is
observed initially at 6–8 weeks in the virgin mouse and increases at
the onset of pregnancy with maximal expression observed at day 16–18
of pregnancy (9, 10, 11). Wnt-5B expression increased 4-fold by day 8 of E
and P treatment of intact mice as compared with the untreated (time
zero) mice and was maximally induced by day 16 (P <
0.004, n = 3) as illustrated in Fig. 4A. Thus, the
increase in Wnt-5B gene expression, which parallels that observed
during midpregnancy, requires chronic E and P treatment. The pattern of
Wnt-5B expression in the ovx mice (Fig. 4B) was similar to that
observed in the intact animal but displayed a more dramatic response.
Wnt-5B expression remained low at day 1 and day 3 but increased 9-fold
at day 14 relative to the day 1 E- and P-treated group
(P < 0.001, n = 3). In contrast to the regulation
of Wnt-2, there was no significant effect of E alone on Wnt-5B
expression in ovx mice. The large increase observed in Wnt-5B
expression in ovx mice may reflect the sensitization of the gland to P
due to the absence of endogenous hormones and a rapid induction of PR
gene expression.
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). Mammary glands of 12-day E-
and P-treated BALB/c mice (Fig. 5H) exhibit a morphology similar to
that observed in an 8-day pregnant mouse. Several differences were
observed, however, between the steroid hormone-treated glands and those
from the normal midpregnant mouse, especially when comparing the first
few days after hormone administration. For example, on the second day
of treatment, transient alveolar proliferation was observed (Fig. 5F).
However, by the fourth day these alveoli were no longer detectable, and
a decrease in the amount of secondary branches was observed (Fig. 5).
This transient alveolar budding has not been reported in mice treated
with pharmacological doses of E and P but is similar to the effect
observed in some strains of mice who respond to ovarian cycling by
producing a transient alveolar proliferation in the mature virgin
gland. This phenomena has, however, not been observed in BALB/c mice
(1, 15). Surprisingly, at day 8 some major ducts displayed a ductal
hypertrophy (Fig. 5G, arrow). This hypertrophy has been
observed in mammary glands of mice implanted with hepatocyte-growth
factor (HGF) and treated with E and P (16). Unlike the alveolar
budding, this ductal hypertrophy was not transient and was still
detectable in some glands at day 12–16 (Fig. 5H). Permanent alveoli
appeared at day 12 and increased in number and density throughout the
remainder of the treatment. This progression of morphological changes
can be compared with the normal gland (1). During pregnancy, alveoli
and secondary branching appear by day 4 and increase in density and
number with the progression of pregnancy (Fig. 5, C and D).
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Differential Regulation of Wnt-2 and Wnt-5B Gene Expression in
PR-/- Mammary Glands and
in PR-/-
Epithelium Transplanted into PR+/+ Stroma after
E and P Treatment
The response of Wnt-2 and Wnt-5B gene expression to the onset of
pregnancy and exogenous E and P suggested that the P-signaling pathway
might play a primary role in regulating Wnt gene expression in the
mammary gland. To examine the role of the PR in Wnt gene regulation,
Wnt gene expression levels were determined in
PR-/- mice (12) after treatment with E and P.
Wnt-5B gene expression did not change in response to E and P treatment
in PR-/- mice (n = 3, Fig. 6A). However, the E and P repression of Wnt-2 expression
was still observed but was not significant until day 8 of hormone
treatment (P < 0.003, n = 3, Fig. 6B). This
E-induced decrease in Wnt-2 gene expression was observed in the absence
of any detectable changes in mammary gland morphology in the
PR-/- mice.
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). To confirm that the low level of
Wnt-5B expression seen in the different samples was due to the response
in the epithelium and not due to absence or degradation of RNA, the
expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also
analyzed by RT-PCR. G3PDH transcripts are readily detectable and
constant throughout the treatment period (data not shown). The same
129SvEv mice containing transplanted PR-/-
epithelium and treated with E and P for 8 days still possessed the
ability to repress Wnt-2 expression when subjected to steroid hormone
treatment (P < 0.013, n = 3, Fig. 6D).
Interestingly, the reduction of Wnt-2 gene expression was again delayed
requiring 8 days of steroid hormone treatment as observed previously in
PR-/- mice. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, D and
E), lobuloalveolar development was observed in response to E and P,
indicating that the absence of the PR in the stroma does not
significantly impair the ability of the epithelium to undergo alveolar
differentiation. However, ductal growth was impaired, as the
PR+/+ epithelium failed to fill the
PR-/- fat pad. In addition to the limited
ductal development, unusual distended endbuds were present in these
outgrowths. This restricted ductal growth may be due to disruption of
reciprocal interactions between the stromal and epithelial compartments
required for this stage of development. Less dramatic effects on ductal
morphogenesis have been observed in the PR-/-
mouse (12). This suggests that the PR-/-
stroma, when recombined with PR+/+ epithelium in
vivo, lacks the mechanism to correctly interact with and stimulate
growth in the epithelium and that the stroma fails to generate signals
upon which the ductal growth of the epithelium is dependent. In
contrast, in the PR-/- mouse, the
PR-/- epithelium, in the presence of
PR-/- stroma, has adapted to the absence of
reciprocal signals, and the epithelium has become independent of these
inputs required for ductal growth. This hormone-dependent reciprocity
of growth regulation has been observed previously in recombination
experiments with wild type and TfM androgen-insensitive mammary glands.
These experiments revealed that the epithelium can induce the
expression of mesenchymal androgen receptors. In turn, the mesenchyme
condenses around the epithelium and causes an epithelial regression
(18, 19). The PR in the stroma is expressed in a temporally distinct pattern from the epithelial receptor, has a different signaling mechanism, and affects a separate group of target genes (5). Therefore, these data support the theory of two separate functional effects of mammary gland PR based on their compartmentalization and roles in development. The unexpected results observed in transplants of PR+/+ epithelium in the PR-/- stroma suggest that there is a P-dependent stromal signal for ductal development. Recent in vivo studies of murine mammary glands treated with HGF in the presence of E and P suggest that HGF is a potential candidate second messenger for ductal growth in the mammary gland (16, 20). HGF-treated mammary glands respond to E and P by stimulating ductal growth. Interestingly, this growth factor has also been shown to regulate Wnt-5A expression (21). If this hypothesis is valid, it may be possible to rescue the PR-/- stroma defect by the direct addition of HGF. Unfortunately, because HGF knockouts are embryonic lethal and die before E16.5, no information has been obtained to date on mammary ductal development in these knockout mice (22).
Wnt-2 and Wnt-5B Gene Expression Are Regulated Independently by
Steroid Hormones
This study demonstrates that two developmentally regulated
Wnt genes are regulated by distinct mechanisms. The unique temporal and
spatial patterns of expression of Wnt-2 and Wnt-5B suggest that these
genes may play some role in the development of the mammary gland. The
response of Wnt-2 and Wnt-5B to E and P treatment indicates that these
genes are useful markers for the action of E in the virgin mammary
gland, and for P during pregnancy, respectively. Wnt-2 gene expression
is highest in the immature virgin gland of BALB/c mice and declines
rapidly at the onset of pregnancy (9). In ovx and intact BALB/c mice
this effect can be mimicked with the addition of pharmacological doses
of E. This acute repression of Wnt-2 gene expression is correlated with
the appearance of lobuloalveolar structures and the termination of
ductal development. Conversely, an increase in Wnt-5B gene expression
in ovx and intact mice requires chronic treatment with E and P.
Ovariectomy enhances the magnitude of this response. Without the
stimulation from the ovaries, the basal levels of Wnt-5B expression are
probably significantly reduced, thereby allowing an enhanced response.
Wnt 5B provides one of the few molecular endpoints for the action of P,
and changes in Wnt 5B are coincident with lobuloalveolar
development.
The results from the PR-/- studies demonstrate that Wnt-5B gene expression is induced by P and dependent on the presence of PR specifically in the epithelial compartment. E alone has no effect on Wnt-5B expression. Interestingly, the PR present in the stroma cannot compensate for the absence of PR in the epithelium for lobuloalveolar development or for induction of Wnt 5B gene expression. This result implies that P acts directly on the epithelium to induce lobuloalveolar development and, either directly or indirectly, to activate Wnt-5B gene expression. E is required for induction and maintenance of PR expression in the mammary gland (6). Therefore, it is unlikely that the regulation of Wnt-5B expression is independent of E.
Wnt-2 gene expression was inhibited by administration of E in both PR-/- mice and in transplanted PR-/- epithelium. These results suggest that Wnt-2 gene expression is not primarily regulated by PR signaling. In the PR-/- studies there was a delay in the kinetics of the Wnt-2 response. The absence of the PR may have restricted the development of the gland in these mice and slowed the appearance of the ER, which normally appears at 4 weeks of age (23, 24). Alternatively, the absence of the PR could influence reciprocal interactions between the stroma and the epithelium, thereby preventing proper induction of ERs. ER gene expression is affected by feedback controls between E and other hormones including P (14).
Previous studies performed in Parks mice demonstrated Wnt-2 expression through midpregnancy and a repression of Wnt-2 and Wnt-5B expression after ovariectomy (11). Parks mice possess virgin lobuloalveolar development, which is absent in BALB/c mice, and it is possible that this epithelial sensitivity to estrous-associated hormones alters the regulation of Wnt-2 and Wnt-5B gene expression.
The rapid repression of Wnt-2 expression suggests the ER may be directly regulating Wnt-2. The ER is expressed in both the stroma and epithelium including the endbud (5, 14, 23, 24, 25). Interestingly, PR is induced by the ER-signaling pathway in the epithelial compartment 48 h after initial addition of E (26). This temporal delay in receptor response coincides exactly with the initial decrease observed in Wnt-2 gene expression after hormonal treatment. Therefore, the timing of this E-induced gene expression and localization of some Wnt-2 transcripts in the epithelium suggests that Wnt-2 could be regulated directly by E.
The Wnts Are Growth Factors with Pleiotropic Effects on
Development
The development of the mammary gland is dependent on the
interaction and cooperation of growth factors and hormones functioning
through the stromal and epithelial compartments. Studies of PRL,
epidermal growth factor, FGF, TGF
and ß, insulin-like growth
factor, and HGF action reveal that they are regulated in specific
spatial and temporal patterns and have effects on proliferation and
differentiation in mammary gland development (1, 27, 28, 29, 30). The
developmentally associated expression pattern, their role in the
development of other organisms, biochemical characteristics, and
hormonal regulation of the Wnts suggest that they are members of this
complex family of locally acting growth factors.
The function of the Wnt genes in the development of the mammary gland can only be inferred from limited expression studies in vivo and in vitro and functional studies in other organisms. Wingless, the Drosophila homolog of Wnt-1, has proliferative, inductive, and cell fate determination functions (8). In addition, Wnt genes have demonstrated functional roles in Xenopus, mouse, and chicken (31, 32, 33, 34, 35, 36). These diverse studies revealed that Wnt genes can possess inductive, growth-stimulatory, and growth-restrictive functions all within a single organism.
In the mammary gland, overexpression of Wnt-1 influences the proliferation of mammary epithelium (37). The expression of Wnt-4 and Wnt-5A has been inversely correlated with proliferation in mammary epithelial cells (38). Because of its localization both within and around the highly proliferative terminal endbud, it is possible that Wnt-2 has a role in regulating proliferation in the virgin gland. The pattern of Wnt-5B expression and its dependence on the PR suggests that it interacts with cells in a more differentiated state. Interestingly, proliferation is high in the pregnant gland coincident with the increase in Wnt-5B expression. Localization of Wnt-5B transcripts to the ductal epithelium reveals it is expressed in the proper cellular location to be involved in regulating proliferation in these cells. The localization of Wnt-5B and Wnt-2 transcripts to the ductal epithelial and stromal compartments of the mammary gland (9, 11), respectively, suggest that although these two genes may have separate or even overlapping functional roles, their temporal and spatial expression patterns restrict their activity to specific stages of development. Therefore, it is probable that the expression of these Wnt genes is regulated in a specific manner to restrict their functional activities to particular developmental stages in the mammary gland.
Alteration of Wnt Gene Expression Can Transform Mammary
Epithelium
In the mammary gland, ectopic expression of the Wnt genes has
dramatic consequences on the transformation and development of the
gland. Inappropriate expression of Wnts either temporally or spatially
may result in mammary tumorigenesis. For example, Wnt-1 and Wnt-4 have
been demonstrated to affect the development and transformation of the
gland in vivo (37, 39, 40). Numerous other Wnts, including
Wnt-2 and Wnt-5B, have in vitro transforming effects (41, 42). These in vitro transfection experiments have revealed
that separate classes of Wnts exist that are distinguished by their
transforming ability (43), although the properties defined in these
in vitro assays do not always correspond to their effects
in vivo (R. C. Humphreys and J. M. Rosen, submitted for
publication).
In addition, overexpression of Wnt genes, including Wnt-2 and Wnt-5B, has been found associated with tumors in the breast and intestinal epithelium (44, 45, 46, 47). Thus, loss of regulatory control on these two Wnt genes, as with other growth factor molecules like TGF-ß and FGF (48, 49), has deleterious consequences for the development of the mammary gland. Interestingly, compartment switching of Wnt-2 expression from breast fibroblasts to tumor epithelium has been observed recently in human breast tumors (50). Therefore, there is evidence for a critical role of Wnt-2, and possibly Wnt-5B, in the transformation of the gland. Since most of the Wnt knockouts are embryonic lethals resulting in neural or kidney defects, the precise functional roles of these and other Wnt family members on normal mammary gland development will require the use of tissue-specific or regulated knockouts.
To summarize, the Wnt genes act in a cell-autonomous manner in cooperation with other growth factors and have pleuripotent effects on various developmental processes within the same organism (8). Wnt gene expression can be differentially regulated by steroid hormones in the mammary stroma and epithelium where they may act as locally acting growth factors to influence ductal and lobuloalveolar development. Hopefully, with the recent discovery of the Wnt receptor in Drosophila (51), the mechanism of Wnt action and the function of the individual Wnt family members in mammary gland development will begin to be illuminated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Isolation of Mammary Glands and RNA
Number 4 (thoracic) mammary glands were removed from aged
matched 6- and 8-week-old virgin BALB/c and C3H mice using standard
surgical techniques. The thoracic and inguinal mammary glands from
11-week-old wild type control mice, 6-week-old 129SvEv
PR-/- mice, 129SvEv PR+/+, and
129SvEv PR-/- mice with transplanted
PR-deficient epithelium or wild type epithelium, respectively, were
removed using standard surgical techniques. For morphological analysis,
mammary glands were fixed in Tellyesniczky’s solution for 5 h and
stained with hematoxylin as described previously (52). For isolation of
RNA, mammary glands were homogenized in a PT2000 Polytron (Brinkmann,
Westbury, NY) with RNazol (Biotecx, Houston, TX) as described by the
manufacturer or homogenized in 4 M guanididium
isothiocynate (Sigma, St. Louis MO) and isolated by CsCl centrifugation
method. RNA was quantitated spectrophotometrically and stored at -20 C
in 70% ethanol.
Construction and Transcription of cRNA Templates
Quantitative noncompetitive RT-PCR was performed as previously
described (9). Complementary DNAs from Wnt-5B and Wnt-2 were prepared
according to standard bacterial plasmid isolation protocols, and the
DNA was purified on Qiagen (Qiagen, Chatsworth, CA) columns according
to the manufacturer and isolated from the vector using unique
restriction enzymes. To construct the Wnt-2 cDNA deletion template,
StyI (New England Biolabs, Beverly, MA) was used to excise a
fragment from bases 493–580. Digestion products were separated from
the small internal fragment, religated, and subcloned into the vector
pBKSII (Stratagene, La Jolla, CA). Clones were analyzed for a size
difference and sequenced to confirm the location of the deletion. The
Wnt-5B template was constructed in the same manner with an
AvaI (New England Biolabs, Beverly MA) deletion of bases
501–576. Both templates were sequenced to confirm the orientation in
the vector and the presence of an internal deletion. These constructs
were used as templates for in vitro transcription reactions
as described in Promega Protocols and Applications Guide, ed 2
(Promega, Madison WI). The cRNA reactions were treated with 1 U of
ribonuclease-free RQ1 deoxyribonuclease in deoxyribonuclease buffer
(Promega) for 60 min at 37 C and then extracted with phenol-chloroform
twice and precipitated with 3 M NaAc and 100% ethanol at
-20 C. The cRNA was resuspended in Tris-EDTA, quantitated
spectrophotometrically, and stored at -20 C in 70% ethanol. Each
template was assayed by PCR to confirm the absence of contaminating
cDNA template. Optimum RT-PCR conditions for each of the templates were
developed that allowed a linear response with respect to the RNA input
and exhibited noncompetitive PCR.
Quantitative RT-PCR
Isolated RNA was transcribed in a reaction consisting of 1x
Taq polymerase buffer (Promega), 3 mM
MgCl2, 100 pmol hexanucleotide random primers (Boehringer
Mannheim, Indianapolis, IN) 1.25 U of RT (GIBCO BRL, Gaithersburg MD),
1 mM of each of four deoxynucleoside triphosphates
(Pharmacia, Milwaukee WI), and 20 U of RNasin (Pharmacia, Milwaukee WI)
in a final reaction volume of 20 µl. Fifty nanograms, 100 ng, and 150
ng of sample RNA were added to separate RT reactions. A constant amount
of cRNA template (
10,000 molecules) was added to each RT reaction as
an internal standard to control for differences in RT and PCR reaction
efficiency.
The primer sequences for the Wnt-2 and Wnt-5B amplifications, respectively, were:
forward: 5'-AGTCGGGAATCGGCCTTTGTTTACG-3' and reverse:
5'-AAAGTTCTTCGCGAAATGTCGGAAG-3'; forward: 5'-GACAGCGCCGCGGCCATGCGC-3'
and reverse: 5'-CATTTGCAGGCGACATCAGC-3'. PCR conditions were 94 C for 1
min, 60 C for 2 min, and 72 C for 3 min, for 30 cycles and 94 C for 1
min, 65 C for 2 min, and 72 C for 3 min for 32 cycles for Wnt-2 and
Wnt-5B, respectively. Primers for G3PDH were: forward:
5'-AGAGGCCTTTGCTCGAACTGGAAAG-3' and reverse:
5'-CACCAAGACGTCTGTCGCCTACTTA-3. PCR conditions were 94 C for 1 min, 60
C for 2 min, and 72 C for 3 min, for 30 cycles. All PCRs were followed
by an extension at 72 C for 5 min. PCR was performed with 10 µl of
each RT reaction, 2 mM magnesium chloride, 1xPCR buffer
(Promega), 0.1 µCi [-32P]dCTP (NEN DuPont,
Boston, MA), 1 U of Taq polymerase (Promega, Madison WI) in
a final reaction volume of 50 µl. Ten microliters of the RT-PCR
products were separated on a 2% Nusieve agarose (FMC Bioproducts,
Rockland, ME) gel and transferred overnight in 0.4 M NaOH
to Hybond N+ nylon membrane (Amersham, Buckinghamshire,
UK), and the radioactive signal was quantitated with 4–8 h exposure on
a PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
Steroid Hormone Treatment of Mice
All groups of mice were treated with 1 mg of P (Steris, Phoenix,
AZ) and 1 µg of 17 ß-estradiol (Sigma) per day in 60 µl of sesame
seed oil (Sigma) subcutaneously. Mammary glands were collected at days
0, 1, 2, 4, 8, 12, and 18. Animals were ovariectomized and allowed to
regress for 4 weeks before hormone treatments were begun. Beeswax
implants containing 20 µg of E and/or 20 mg of P were synthesized by
adding the powdered form of the hormones to melted beeswax. The
suspended hormone mixture was dropped onto dry ice to form pellets. The
pellets, synthesized to deliver 1 mg P and 1 µg E/day, respectively,
were implanted subcutaneously in the neck of the mice for 2 weeks.
Inguinal mammary glands were collected at days 1, 3, and 14 and
analyzed as described.
Transplantation Studies
Tissue fragments of 10-week-old virgin
PR-/- mammary epithelium were isolated and
implanted into six PR-positive 129SvEv hosts using the technique
described by DeOme et al. (13). Epithelium from 129SvEv
hosts was removed as described (13). In addition, tissue fragments from
a 10-day pregnant 129SvEv PR+/+ mammary epithelium were
isolated and implanted into four PR-/-
129SvEv hosts. Due to the limited number of
PR-/- homozygote recipients and the limited
extent of ductal outgrowth, these glands could not be examined for
changes in Wnt gene expression. Mammary gland epithelial transplants
were allowed to proliferate and penetrate the stromal fat pad for 10
weeks and then treated with steroid hormones as described above.
Control experiments with wild type 129SvEv PR+/+ epithelium
into cleared fat pads of three wild type 129SvEv PR+/+ mice
were performed in the same manner.
Whole Mount Staining and Sectioning
The whole gland staining was carried out essentially as
described (25) except that glands were stained for only 2 h in
hematoxylin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant CA-64255 and Grant DAMD17-94-J-4253 from the Department of Defense (to J.M.R.).
Received for publication September 9, 1996. Revision received November 14, 1996. Accepted for publication November 21, 1996.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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K.-U. Wagner, A. Krempler, A. A. Triplett, Y. Qi, N. M. George, J. Zhu, and H. Rui Impaired Alveologenesis and Maintenance of Secretory Mammary Epithelial Cells in Jak2 Conditional Knockout Mice Mol. Cell. Biol., June 15, 2004; 24(12): 5510 - 5520. [Abstract] [Full Text] [PDF]
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 C. J. Ormandy, M. Naylor, J. Harris, F. Robertson, N. D. Horseman, G. J. Lindeman, J. Visvader, and P. A. Kelly Investigation of the Transcriptional Changes Underlying Functional Defects in the Mammary Glands of Prolactin Receptor Knockout Mice Recent Prog. Horm. Res., January 1, 2003; 58(1): 297 - 323. [Abstract] [Full Text] [PDF]
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M. Jakacka, M. Ito, F. Martinson, T. Ishikawa, E. J. Lee, and J. L. Jameson An Estrogen Receptor (ER){alpha} Deoxyribonucleic Acid-Binding Domain Knock-In Mutation Provides Evidence for Nonclassical ER Pathway Signaling in Vivo Mol. Endocrinol., October 1, 2002; 16(10): 2188 - 2201. [Abstract] [Full Text] [PDF]
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G. S. Prins, W. Y. Chang, Y. Wang, and R. B. van Breemen Retinoic Acid Receptors and Retinoids Are Up-Regulated in the Developing and Adult Rat Prostate by Neonatal Estrogen Exposure Endocrinology, September 1, 2002; 143(9): 3628 - 3640. [Abstract] [Full Text] [PDF]
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M. Kingsley-Kallesen, S. S. Mukhopadhyay, S. L. Wyszomierski, S. Schanler, G. Schutz, and J. M. Rosen The Mineralocorticoid Receptor May Compensate for the Loss of the Glucocorticoid Receptor at Specific Stages of Mammary Gland Development Mol. Endocrinol., September 1, 2002; 16(9): 2008 - 2018. [Abstract] [Full Text] [PDF]
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J. M. Shillingford, K. Miyoshi, M. Flagella, G. E. Shull, and L. Hennighausen Mouse Mammary Epithelial Cells Express the Na-K-Cl Cotransporter, NKCC1: Characterization, Localization, and Involvement in Ductal Development and Morphogenesis Mol. Endocrinol., June 1, 2002; 16(6): 1309 - 1321. [Abstract] [Full Text] [PDF]
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S. O. Mueller, J. A. Clark, P. H. Myers, and K. S. Korach Mammary Gland Development in Adult Mice Requires Epithelial and Stromal Estrogen Receptor {alpha} Endocrinology, June 1, 2002; 143(6): 2357 - 2365. [Abstract] [Full Text] [PDF]
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C. L. Smith, D. G. DeVera, D. J. Lamb, Z. Nawaz, Y.-H. Jiang, A. L. Beaudet, and B. W. O'Malley Genetic Ablation of the Steroid Receptor Coactivator-Ubiquitin Ligase, E6-AP, Results in Tissue-Selective Steroid Hormone Resistance and Defects in Reproduction Mol. Cell. Biol., January 15, 2002; 22(2): 525 - 535. [Abstract] [Full Text] [PDF]
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 G. Chakravarty, D. Roy, M. Gonzales, J. Gay, A. Contreras, and J. M. Rosen p190-B, a Rho-GTPase-activating Protein, Is Differentially Expressed in Terminal End Buds and Breast Cancer Cell Growth Differ., July 1, 2000; 11(7): 343 - 354. [Abstract] [Full Text]
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 C. Brisken, A. Heineman, T. Chavarria, B. Elenbaas, J. Tan, S. K. Dey, J. A. McMahon, A. P. McMahon, and R. A. Weinberg Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling Genes & Dev., March 15, 2000; 14(6): 650 - 654. [Abstract] [Full Text]
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 S. Saji, E. V. Jensen, S. Nilsson, T. Rylander, M. Warner, and J.-A. Gustafsson Estrogen receptors alpha and beta in the rodent mammary gland PNAS, January 4, 2000; 97(1): 337 - 342. [Abstract] [Full Text] [PDF]
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J. P. Lydon, G. Ge, F. S. Kittrell, D. Medina, and B. W. O'Malley Murine Mammary Gland Carcinogenesis Is Critically Dependent on Progesterone Receptor Function Cancer Res., September 1, 1999; 59(17): 4276 - 4284. [Abstract] [Full Text] [PDF]
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G. W. Robinson, P. F. Johnson, L. Hennighausen, and E. Sterneck The C/EBPbeta transcription factor regulates epithelial cell proliferation and differentiation in the mammary gland Genes & Dev., June 15, 1998; 12(12): 1907 - 1916. [Abstract] [Full Text]
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C. Brisken, S. Park, T. Vass, J. P. Lydon, B. W. O'Malley, and R. A. Weinberg A paracrine role for the epithelial progesterone receptor in mammary gland development PNAS, April 28, 1998; 95(9): 5076 - 5081. [Abstract] [Full Text] [PDF]
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L. Hennighausen and G. W. Robinson Think globally, act locally: the making of a mouse mammary gland Genes & Dev., February 15, 1998; 12(4): 449 - 455. [Full Text]
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T. K. Said, O. M. Conneely, D. Medina, B. W. O'Malley, and J. P. Lydon Progesterone, in Addition to Estrogen, Induces Cyclin D1 Expression in the Murine Mammary Epithelial Cell, in Vivo Endocrinology, September 1, 1997; 138(9): 3933 - 3939. [Abstract] [Full Text] [PDF]
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