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C 2002)
Journal of Mammary Gland Biology and Neoplasia, Vol. 7, No. 1, January 2002 (!
Establishing a Framework for the Functional Mammary
Gland: From Endocrinology to Morphology
Russell C. Hovey,1 Josephine F. Trott,1 and Barbara K. Vonderhaar1,2
From its embryonic origins, the mammary gland in females undergoes a course of ductal development that supports the establishment of alveolar structures during pregnancy prior to
the onset of lactogenesis. This development includes multiple stages of proliferation and morphogenesis that are largely directed by concurrent alterations in key hormones and growth
factors across various reproductive states. Ductal elongation is directed by estrogen, growth
hormone, insulin-like growth factor-I, and epidermal growth factor, whereas ductal branching
and alveolar budding is influenced by additional factors such as progesterone, prolactin, and
thyroid hormone. The response by the ductal epithelium to various hormones and growth
factors is influenced by epithelial–stromal interactions that differ between species, possibly
directing species-specific morphogenesis. Evolving technologies continue to provide the opportunity to further delineate the regulation of ductal development. Defining the hormonal
control of ductal development should facilitate a better understanding of the mechanisms
underlying mammary gland tumorigenesis.
KEY WORDS: ductal; hormones; growth factors; epithelial–stromal; morphogenesis.
INTRODUCTION
our knowledge identified. Furthermore, similarities in
the morphological development of mammary glands
in humans and ruminants will be indicated, highlighting the potential utility of the latter species as valuable
models for understanding human breast development
and cancer.
During its development the mammary gland progresses through distinct stages: the embryonic and
fetal period when the mammary anlage develops,
the neonatal and prepubertal periods of isometric
growth, the peripubertal period when the gland grows
allometrically and ducts elongate and branch, and
sexual maturity when branching continues and alveolar buds form. All of these stages are covered in
this review and contribute to the basic morphological structure of the mammary gland. The final
stage involves the process of functional differentiation during pregnancy-associated lobuloalveolar development to support lactation, followed by involution when nursing ceases (see chapters by Brisken
et al. and Neville et al.). All of these processes are
hormonally-regulated throughout development.
Development of the mammary gland is influenced by numerous factors, principal among which
are endocrine hormones that interplay with the actions of various growth factors and the epithelial and
mesenchymal constituents. This review will explore
endocrinological aspects of mammary gland development in rodents, ruminants, and humans. While the
rodent mammary gland is the most widely studied and
has provided many biological insights, it does not fully
represent the mammary glands of all species, particularly those of humans. The relevance of the mouse
as a model for normal human mammary gland development will be discussed, and significant gaps in
1
Molecular and Cellular Endocrinology Section, Basic Research
Laboratory, Center for Cancer Research, NCI, National Institutes
of Health, Bethesda, Maryland.
2 To whom correspondence should be addressed at Molecular
and Cellular Endocrinology Section, Building 10, Room 5B47,
10 Center Drive, National Institutes of Health, Bethesda,
Maryland 20892-1402; e-mail: bv10w@nih.gov.
17
C 2002 Plenum Publishing Corporation
1083-3021/02/0100-0017/0 !
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Investigations into the endocrinology of mammary gland growth, differentiation, and tumorigenesis began over 100 years ago when, in 1896,
Beatson (1) removed the ovaries from a breast cancer patient and observed a palliative effect on the
disease. Investigations early last century utilized systematic studies of organ ablation and hormone replacement in rodents and ruminants. These classical studies laid the foundation for much of what
we now know concerning the action of hormones—
agents produced in one organ that are secreted into
the blood and subsequently act on another organ.
These studies showed that estrogen (E),3 progesterone (P), prolactin (PRL), growth hormone (GH),
and thyroid hormones are essential for ductal elongation, branching, and alveolar budding, and that
PRL, adrenal steroids, GH, thyroid hormones, oxytocin, and insulin are required for complete lobuloalveolar development and milk synthesis, secretion,
and lactation. Some of these hormones (E, P, PRL,
and GH) appear to be inductive while others play a
more permissive role. The subsequent development of
various culture systems, transplantation techniques,
and transgenic and knockout (KO) models has refined our understanding of the endocrinological regulation of mammary gland development whereby hormones induce expression of various growth factors
that may function as endocrine or locally acting autocrine/paracrine agents.
EMBRYONIC DEVELOPMENT
AND MORPHOGENESIS
In all mammals the mammary glands arise from
a localized thickening of the ectoderm or epidermis.
The mammary bud forms by elevation of an epidermal
“mammary crest” and a milk-line that forms on both
sides of the midventral line in the embryo. This pattern
of organogenesis is similar in rodents, ruminants, and
humans.
3
Abbreviations: embryonic day (e); estrogen (E); epidermal
growth factor (EGF); epidermal growth factor receptor (EGFR);
estrogen receptor (ER); follicle stimulating hormone (FSH);
growth hormone (GH); hepatocyte growth factor/scatter factor (HGF/SF); insulin-like growth factor (IGF); knockout (KO);
luteinizing hormone (LH); mammary epithelial cell (MEC); mammary fat pad (MFP); progesterone (P); placental lactogen (PL);
progesterone receptor (PR); prolactin (PRL); prolactin receptor (PRLR); parathyroid hormone (PTH); parathyroid hormonerelated protein (PTHrP); terminal duct lobular unit (TDLU);
terminal end bud (TEB); transforming growth factor (TGF).
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Rodents
Development of the mammary gland is essentially the same in the mouse and rat fetus (2). In
the mouse, mammary buds form between embryonic
days 10 (e10) and 11 (e11). Between e11 and e16 there
is little cell proliferation; however, by e13 each bud has
increased in size due to cell migration that leads to a
concentration of epithelial cells within the epidermis.
Additional cells migrate from the adjoining epidermis
to form a connection between the bud and epidermis
that results in the formation of a rudiment at e14. In
females, proliferation from e16 to birth (at e21) leads
to the formation of a small ductal tree consisting of
as many as 15 canalized branches arising from a single duct attached to each nipple (3). Formation of
this anlage from the e13 primordia is independent of
systemic influence since it occurs in culture without
supplemental hormones or growth factors (4).
In the rodent fetus, the mesenchyme of the
mammary gland consists of two distinct compartments (5,6). By e14, the first and slightly denser
mesenchyme orients around the epithelial buds and
comprises several concentric layers of fibroblasts
that arise from the dermis. Thereafter, a less-dense
mesenchyme composed of preadipocytes destined to
become the mammary fat pad (MFP) begins to proliferate on days e16–e17 (5). As the primary sprout of
epithelium grows, it pushes through the dense mesenchymal sheath and penetrates into the primitive
MFP. Lipid begins to accumulate in the MFP beginning at e16; so that the MFP is a distinct depot of white
adipose tissue at birth (7). This MFP subsequently
supports ductal morphogenesis in the late fetal period and throughout postnatal life.
Ruminants
The pattern of development in ovine and bovine
mammary glands is similar (8,9). In the bovine fetus,
four mammary buds ultimately give rise to the four
glands of the udder and first appear when the fetus
is 4–8 cm. The mammary cord becomes canalized to
form the streak canal and cistern at the 19 cm stage
while secondary branches arise from the dilated cistern at the 16–23 cm stage (9). A definitive MFP is
first evident in the bovine fetus around e80 (10). The
mammary glands of fetal male sheep grow constantly
at a rate of 2.8 times that of body weight while the
glands in females grow 5 times faster than the body between e44 and e70 (11). By e70, secondary ducts have
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developed, the teat cistern is evident, and parenchymal tissue is well developed. Mammary growth then
declines to 1.7 times the rate of body growth until
birth (11).
Humans
In the human fetus, mammary epithelial cells
(MEC) arise from an ectodermal bud (12) to form
clusters that later represent the areolus of each breast.
The milk streak is first observed during the 4th week
of embryonic life and becomes the milk line during the 5th and 6th weeks. Between the 7th and
8th weeks the mammary parenchyma begins to invade the underlying stroma and the mammary disc
appears. Further inward growth of the mammary
parenchyma commences at the 9th week, concomitant
with the regression of the overlying skin. Between the
10th and 12th weeks, epithelial buds sprout from the
invading parenchyma, followed by indentation during the 12th and 13th weeks that results in the formation of epithelial buds with notches at the epithelial–
stromal border. Branching of the parenchyma during
the 13th–20th week results in 15–25 epithelial strips
or solid cords that eventually give rise to the multiple openings (galactophores) at each nipple. During the branching process, and up to the 32nd week,
the solid cords become canalized by apoptosis of the
central epithelial cells. Finally, between the 32nd and
40th weeks of gestation, limited lobulo-alveolar development occurs in association with the development
of end vesicles composed of an epithelial monolayer.
In the 32-week-old fetus the periductal stroma has a
loose appearance, while in full-term infants the rudimentary lobular structures are surrounded by a dense
stroma (13).
NEONATAL AND PREPUBERTAL
DEVELOPMENT AND MORPHOGENESIS
The mammary glands of newborns contain only
rudimentary ducts with small club-like ends that
regress within a short time after birth. In neonates of
most species, the mammary gland grows isometrically
before the onset of puberty. This period of growth has
not been extensively studied, particularly in rodents.
The mammary gland of a newborn ruminant
is composed of a teat, a primary duct, and several secondary ducts that end in modestly-branched
lobules (10,14) positioned at the periphery of the
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MFP (15–17). In the neonatal female bovine (heifer),
the parenchyma initially grows isometrically relative
to overall body development, but at about 3 months of
age, prior to puberty, growth becomes allometric (18).
The parenchyma of the prepubertal bovine mammary
gland shows a more complex and branched ductal
structure in cross section than that of the rodent (19).
In children, the mammary glands do little more
than keep pace with the general growth of the body
until the approach of puberty at 8–12 years. At this
stage the female breast begins to show growth activity both in the epithelium and the surrounding
stroma (12).
ENDOCRINOLOGY OF EMBRYONIC,
NEONATAL, AND PREPUBERTAL
MORPHOGENESIS
Endocrine effects on the mammary gland begin during embryonic development and continue
throughout postnatal life. Effects of hormones arise
from changes in serum levels and in the amount and
location of their cognate receptors.
Sexual Dimorphism
In mice and rats, sexual dimorphism is established in utero. Transcription of androgen receptors and estrogen receptors (ER) in the stroma,
induced by the adjacent epithelium, is elevated
at e12 (20,21) concomitant with the expression of
parathyroid hormone-related protein (PTHrP) and
its receptors. The ability to induce steroid receptors in
primary mammary mesenchyme is a capacity unique
to the embryonic mammary epithelium (22).
During embryogenesis, beginning at e12, the
mammary epithelium expresses PTHrP while the
surrounding mesenchyme expresses receptors for
PTH/PTHrP (23). The paracrine stimulation of mesenchymal cells by epithelium-derived PTHrP induces
the formation of the dense mammary mesenchyme
that surrounds the mammary bud. These mammaryspecific mesenchymal cells respond to PTHrP and,
in turn, induce the MEC to migrate into the MFP.
The epithelial–mesenchymal interactions induced by
PTHrP trigger epithelial morphogenesis and stimulate the overlying epidermis to form the nipple (24).
PTHrP action in both the epidermis and mesenchyme
involves interaction with the Wnt signaling pathway (24). PTHrP and its receptors are expressed in
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the breast of normal 16-week-old human fetuses when
it consists of an epithelial bud extending from the
epidermis into the subepidermal mesenchyme (25).
Human fetuses with Blomstrand chondrodysplasia
and an associated lack of PTHR-1 also lack nipples
and breasts.
The mammary gland in rodents at e14 is responsive to E given that exogenous E stimulates
precocious nipple development (26). However, removal of endogenous estrogens by X-irradiation of
the ovaries at e13 was without effect on mammary
development (27), suggesting that the female form is
the default state. This has recently been confirmed in
that glands from female ERKO mice lacking ER-α
develop normally until puberty (28).
In the fetal mouse, testosterone is secreted by the
testes and acts on mesenchymal androgen receptors to
induce stromal condensation around the stalk of the
epithelial bud during a critical 2-day period between
e13.5 and e15.5 (21,29). Induction of the androgen receptor depends on signaling between PTHrP and its
receptors (30). As a result of extensive necrosis, the
epithelial rudiment in the male becomes isolated from
the epidermis and in many cases undergoes complete
regression (2,31). The penetrance of this phenotype
varies with mouse strains (G. H. Smith, personal communication). In those male mice that retain a mammary rudiment, it remains dormant throughout life.
However, exogenous E, in the presence or absence
of P, stimulates ductal outgrowth and milk protein
synthesis (31). In rats, however, the mammary anlage
is retained in most MFPs and is capable of extensive
proliferation, but has no outlet to the nipple (32).
Sexual dimorphism within the mammary glands
of ruminants is not evident until about 3 months of
age, prior to puberty (19). In humans there is no
evidence of sexual dimorphism until the onset of
puberty (12).
resulting from exposure to maternal sex steroids. In
addition, this tissue synthesizes milk proteins when
cultured in the presence of lactogenic hormones, or
transplanted into cleared MFPs or under the kidney
capsule of hormone-treated nulliparous or pregnant
mice (29,33,34). Our recent data (35) has shown that
mRNA for all isoforms of the PRLR is expressed in
both MEC and the MFP in newborn mice prior to puberty (35). Expression of mRNA for the short forms
of the PRLR within the MFP decreases after birth
in an isoform-specific pattern to very low levels during puberty, and beyond. In rats and mice, production of PRL by the pituitary does not commence until
birth (36). The predominant lactogen during mid- to
late gestation in rodents appears to be placental lactogen (PL) II, seen as early as e17 in rats (37) and e16
in mice (38). Both PL and PRL bind to the PRLR in
the mammary glands. Whether PL has direct effects
on the mammary glands of the fetus is unknown. The
contribution of PRL to the development of mammary
glands in neonatal mice remains to be determined.
In the mouse, ER can be demonstrated at low
levels in both MEC and the MFP as early as the third
day post-partum (39). Immunohistochemical staining
for ER in MEC increases in the intensity and number of positive cells (from 8% to 20%) between one
week of age and puberty. Only 4% of stromal cells
are weakly positive for ER at day 3; however, the
number of positive cells and staining intensity continue to increase through puberty. Staining in the
stroma is confined to undifferentiated mesenchymal
cells rather than adipocytes or fibroblasts, and is not
locally concentrated near the epithelium. Despite this
presence of ER, E does not induce the binding of P
in the mammary gland until 7 weeks of age (40). We
recently showed that mRNA for the progesterone receptor (PR) is undetectable in the mouse mammary
gland until 3 weeks of age (35).
Responsiveness to Lactogenic and Sex
Steroid Hormones
Ruminants
Rodents
The mammary glands of fetal and neonatal rodents and humans contain functional receptors for
a variety of hormones, thus rendering them responsive to maternal steroid hormones and lactogens both
in vivo and in vitro. In the late-fetal and newborn
mouse there is a transient appearance of terminal end
buds (TEB) at the end of the primitive ducts, possibly
Mammary parenchyma in prepubertal heifers
primed with E and P only undergoes functional differentiation in response to lactogenic stimulation after it acquires a specific developmental state (41).
Upon acquisition of this state, sexually immature, prepubertal Holstein heifers can be induced to lactate
by treatment with E and P followed by dexamethasone and hand milking (42). In addition, explants of
mammary parenchyma from immature bulls primed
with E and P can synthesize and secrete milk proteins
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when cultured in the presence of a lactogenic hormone mix (43). Taken together, these data indicate
that receptors for these hormones are present and active in the mammary gland of the prepubertal bovine.
Humans
During the last stages of gestation, the distal
portion of the mammary ducts develop into alveolar
structures and MEC appear secretory. At birth the
distended ducts contain milk-like secretion that can
be expressed. Known as witch’s milk, this secretion
can be observed in most infants by the first week of
postnatal life, and lasts for up to 6 weeks. GH and hPL,
both of which bind to the hPRLR with high affinity,
are present in human fetal serum as early as 8 weeks
of pregnancy, reaching a level of 35–500 ng/mL at
midgestation. Serum PRL is low (10–20 ng/mL) until the beginning of the third trimester, after which
time fetal PRL rises to peak at about 150 ng/mL at
term (44). Once the effects of placental and maternal
hormones subside in newborn children, the secretory
alveoli regress so that only scattered ducts without
alveoli lie embedded in stromal tissue (12).
ER and PR are present in breast MEC in human fetuses and infants. By immunohistochemistry,
ER-α was detected in 5% of MEC beginning at the
30th week of gestation (45), where ER expression associated with high levels of MEC proliferation (13).
ER levels increased during the remainder of gestation
and increased markedly shortly after birth. Thereafter, PR was expressed in 5 to 60% of MEC for up
to 3 months.
Taken together, these observations indicate the
presence of functional receptors for E, P, and PRL in
mammary glands of the fetus and neonate in rodents,
ruminants, and humans. The action of these hormones
in a variety of systems has been shown to result from
induction of autocrine/paracrine growth factors such
as epidermal growth factor (EGF) and transforming
growth factor-α (TGF-α). In the mammary glands of
the fetus and neonate, hormonal induction of such
growth factors has not been directly established. However, the EGF receptor (EGFR) has been identified
in a variety of cell types in the developing mouse
mammary gland, implicating EGF as a mediator of
epithelial–stromal interactions. The EGFR is not essential for development of the mammary anlage since
EGFRKO mice have normal ductal development at
birth. However, the ducts in mammary glands from
11-day-old female EGFRKO mice infiltrate into the
21
MFP much less than in wild type mice (46), suggesting a role for EGF or its relatives during isometric
growth of the mammary rudiment. In the breasts of
human fetuses, weak TGF-α immunoreactivity localizes to the developing stroma and epithelial bud (47).
In the infant, TGF-α positive cells are generally more
numerous in end buds and lobular buds. The function
of TGF-α in the breasts of either the fetus or neonate
is unknown, although areas of TGF-α expression are
often associated with increased vascularity of the adjacent mesenchyme.
Inappropriate Hormonal Exposure
Various studies have underlined the importance
of inappropriate hormonal exposure during the fetal
and neonatal periods. The neonatal mammary gland
in the mouse is comparable to the embryonic mammary gland in humans. Inappropriate exposure of
neonatal female rodents to estrogens, environmental endocrine disrupters, androgens, or PRL increases
the sensitivity of the gland to mammotrophic hormones in adulthood. This leads to aberrant ductal
growth and differentiation (48). An interesting observation in mice neonatally exposed to the synthetic E
(diethylstilbestrol) is the precocious appearance of
milk proteins in nulliparous females. These changes
coincide with the synthesis of autocrine PRL within
the mammary gland.4 Such data highlight an interesting facet of endocrinological regulation during mammary gland development that is only now unfolding—
that the mammary gland makes significant amounts of
hormones. Aromatase within the MFP synthesizes E
(49) while MECs make PRL (50) and GH (51).
It remains to be determined what other hormones
are made by the mammary gland. In utero exposure to estrogens in humans may influence breast
cancer incidence (52). Along this line, serum levels
of E vary widely between individuals during pregnancy, prompting Trichopoulos (53) to hypothesize
that elevated maternal E increases the probability
of breast cancer in daughters. Indeed, low E levels
characteristic of preeclampsia/eclampsia are correlated with reduced breast cancer incidence in female
offspring, whereas increased birth weight correlates
4
R. C. Hovey,∗ M. Asai,∗ A. Warri, B. Terry-Koroma, N. Colyn,
E. Ginsburg, and B. K. Vonderhaar. Effects of neonatal exposure
to diethylstilbestrol, tamoxifen and toremifene on the BALB/c
mouse mammary gland. I. Morphological and biochemical responses. Submitted, 2001.
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with high E levels during gestation and is associated
with increased breast cancer incidence (54). Increased
mass of the placenta (the main E producing organ
during pregnancy) is also associated with increased
breast cancer risk (55). The potential significance of
these correlations is suggested by studies with rodents
showing that exposure to diethylstilbestrol both in
utero and neonatally leads to increased susceptibility of the mammary glands to carcinogens (48,56).
ALLOMETRIC GROWTH
Morphogenesis and Development
Rodents
After its course of isometric growth, the mammary gland undergoes a period of allometric growth
to establish the ductal network prior to pregnancy. Allometric growth is defined relative to metabolic body
weight (a function of body weight {BW2/3 } that is
proportional to a growing animal’s surface area) and
can be described by the allometric equation y = bx χ ,
where y = total mammary gland area, x = body
weight2/3 , and b and χ are constants. Hence, x = 1
during isometric growth, whereas x > 1 during positive allometric growth (57). Allometric growth commences at around 31 days of age in the mouse (58) and
around 23 days of age in the rat (59), concurrent with
the commencement of ovarian function and the initiation of puberty. Precisely when puberty commences
in female mice depends on the parameters measured
and the strain under study. The first events of puberty
are increases in circulating gonadatropin and estrogen
levels between 23 and 35 days of age that coincide
with vaginal opening, followed by vaginal cornification several days later (60). Complete cyclicity then
commences approximately 2 weeks after the first detection of vaginal cornification, although the timing
of this onset is strain-dependent (60). Following the
onset of puberty, mammary gland area in rats (59) and
mice (61) increases at 3.5 and 5 times that of metabolic
weight gain until 40 and 56 days of age, and plateaus
by 100 and 110 days of age, respectively.
Histomorphological changes during allometric
development of the mammary gland have been most
extensively characterized in the mouse. At around
26 days of age, duct terminii become enlarged and
acquire a bulbous form referred to as the terminal
end bud (TEB). The TEB is unique in its form and
represents a major site of mitosis that facilitates ductal
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elongation and ramification into the MFP. TEB vary in
their shape and size, having a diameter of 0.1–0.5 mm,
and are largest on the peripheral duct ends. The distal, outermost layer of the TEB is composed of a single layer of pale-staining cap cells that lack polarity
and an organized cytoskeleton, and are only loosely
adherent with each other (Fig. 1(A) and (B)). It has
been proposed that these cells constitute a pluripotent
population that gives rise to multiple cell types (62).
As the duct advances, cap cells may reposition along
the perimeter of the TEB and acquire characteristics
of myoepithelial cells. Other cap cells migrate inward
toward the lumen to become body cells that subsequently constitute the ductal epithelium. While body
cells of the TEB undergo extensive levels of mitosis
(Fig. 1(G)), this population also undergoes an extensive amount of concurrent apoptosis (Fig. 1(H); 64).
In female mice, the TEB continues to facilitate ductal
elongation during puberty (Fig. 2(A)) until the ductal
tree reaches the bounds of the MFP at approximately
8 weeks of age, depending on the strain. During elongation, ducts also form branches that fill the intervening spaces of the MFP as directed by TEBs. Interestingly, however, ducts never come within 250 µm of
each other (66), most likely because of the diffusion
of local inhibitory signals from adjacent ducts.
Ruminants
Allometric mammary growth in ruminants
clearly commences prior to the onset of puberty. The
distinctive timing of this onset relative to mice and
humans can likely be explained by the fact that puberty in ruminants is considered to commence at the
time of first-detectable cyclicity rather than in association with earlier hormonal changes. However, the
associated histomorphological changes during this period in ruminants have not been extensively characterized. A single gland cistern arises from the primary
duct and extends from the teat, giving rise to multiple
ducts. Each duct is surrounded by numerous ductules,
so that in cross section the parenchyma is composed
of many ductal units (Fig. 2(B)). This histomorphogenesis remains evident throughout allometric development and is remarkably similar to that reported
in terminal duct lobular unit (TDLU) structures
within the normal human breast (Fig. 2(C); 12,16).
Somewhat consistent with observations in the human
breast, the majority of proliferation in ruminant mammary parenchyma appears to occur in ductules of
the TDLU. Indeed, Ellis et al. (67) were unable to
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Fig. 1. Terminal end bud (TEB) histology, development, and hormone receptor expression in the
mouse mammary gland. (A) Histology of a TEB found at estrus in an 8-week-old BALB/c mouse;
(B) Schematic diagram of the various cell types in a typical TEB (reprinted with permission; 62);
C) Histology of alveolar buds and tertiary branch points found at diestrus in an 8-week-old
BALB/c mouse; (D) Immunohistochemical localization of ER-α in a transverse section of a TEB
(reprinted with permission; 63). Dark cells are stained positive for ER-α. Cells overlain with silver
grains are undergoing DNA synthesis and are different from those expressing ER-α; (E) Distribution of PRLR mRNA in a TEB as detected by in situ hybridization (reprinted with permission; 35);
F) Distribution of PR mRNA in a TEB as detected by in situ hybridization (reprinted with
permission; 35); G) Cell proliferation within a TEB as shown by the presence of dark-stained
BrdU-labeled cells (courtesy of Robin Humphreys, NIDDK, NIH); (H) Apoptosis within a TEB
demonstrated by dark-stained cells that have been identified by end-labeling (courtesy of Robin
Humphreys, NIDDK, NIH). a, adipocyte; bd, body cell; bl, basal lamina; cp, cap cell; f, fibroblast;
mc, myoepithelial cell.
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liveweight from 2–3 months until about 9 months
of age, and then grows isometrically between 9 and
16 months of age (18). A similar phase of allometric growth occurs in sheep between approximately
8 and 20 weeks of age (17,70–72). It is of considerable noteworthiness that excess dietary energy fed to
ruminants during this period can impair mammary
gland development and subsequent lactational performance (73).
Humans
Epithelial proliferation within the human breast
commences at the onset of puberty in association with
a marked increase in its size due to lipid accumulation
within the MFP (12). At this time the site of active
proliferation is a TEB-like structure, although compared to that in the mouse, it is not as bulbous and
appears to lack some of the same dominant histological features. Following the onset of puberty the
ductal tree elongates under the direction of TEBlike structures and simultaneously undergoes considerable sympodial branching (74). During continued
allometric growth after the first menses, a terminal
duct may give rise to an average of 11 surrounding
alveolar buds, leading to the formation of a TDLU,
type 1 (Fig. 2(C)). Based on cell proliferation indices,
highest proliferation in the human breast is found in
MEC of TDLU, type 1. These structures subsequently
develop into TDLU types 2 and 3 with recurrent menstrual cycles (see below).
Endocrinology of Allometric Growth
and Morphogenesis
Fig. 2. Species comparison of ductal morphogenesis. (A) Ductal
outgrowth in the mammary gland of a peripubescent 8-week-old
BALB/c mouse; (B) TDLU within the mammary gland of a prepubertal 6-week-old ewe lamb; (C) TDLU type 1 within the breast
of a nulliparous 18-year-old human female (reprinted with permission; 65).
identify TEB structures in the ovine mammary gland,
although there clearly are zones of proliferation,
given that mitosis is greatest at the periphery of
the mammary parenchyma (68,69). Likewise, cell
division in ductal MEC of prepubertal heifers occurs randomly rather than being concentrated in an
active growing site (68). In heifers, the mammary
gland increases in size 3.5 times faster than metabolic
Rodents
The hormonal cues that signal the onset of allometric growth may vary between species. In mice,
it is primarily the onset of ovarian function and
estrogen secretion that initiates allometric growth.
Prepubertal ovariectomy completely halts ductal development and leads to the regression of any TEB
structures. Ovariectomy-abrogated ductal growth can
be restored by moderate levels of exogenous E (61),
while high doses suppress ductal growth (75). The critical function of E during ductal growth is confirmed
by the fact that ERKO mice do not undergo normal ductal development (28). It is also clear that E
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acts directly on the mammary gland (76). Despite this
fact, there are only minor changes in serum E levels
during this period (58), suggesting that additional
factors may contribute to the initiation of allometric growth. In the mouse, ER are absent from cap
cells, adipocytes, and myoepithelial cells during ductal elongation; while undifferentiated stromal fibroblasts and luminal MEC in the end bud and ducts
are ER positive (Fig. 1(D); 77). The proportion of
ER-positive cells is highest in the epithelium at 7–
10 weeks, and in the stroma at 6–7 weeks. It appears that E stimulates ductal development indirectly
through stromal ER, despite the presence of ER in
epithelial and stromal cell populations. Evidence for
this paracrine mechanism stems from the demonstration that exogenous E stimulates stromal proliferation in advance of epithelial proliferation (78) and
that stromal ER appears to be required for the development of ducts from MEC derived from neonates,
as determined from heterologous transplantation
experiments (79).
While ductal elongation in the mouse requires E,
it does not proceed in the absence of the pituitary.
Early studies by workers such as Flux (61) demonstrated that a combination of GH plus E was more effective in stimulating ductal development than either
hormone alone. In addition, workers such as Lyons
and Nandi showed that full ductal development in
ovariectomized, adrenalectomized, and hypophysectomized rats and mice requires the combined presence of E, GH, and adrenal corticoid (reviewed
in Ref. 2). These findings have been reiterated more
recently by the demonstration that the mammary
glands of mice lacking the GH receptor fail to undergo ductal elongation (80). Furthermore, it is clear
that insulin-like growth factor-I (IGF-I) functions as
a local effector of GH action, in cooperation with
E. Exogenous GH, but not PRL, induces expression
of IGF-I in the MFP (81), a response that is potentiated by E, and that is aided by the induction of
ER by GH (82). Stroma-derived IGF-I then likely
acts via IGF-I receptors on MEC to stimulate their
proliferation (83) in cooperation with the effects of
E (81). In keeping with this proposal, MECs lacking the IGF-I receptor fail to undergo normal ductal
elongation (84).
It is generally accepted that E and GH are the
principal hormones responsible for TEB formation
and ductal proliferation in the mouse. However, it is
conceivable that other systemic hormones also contribute to ductal elongation. Along these lines, mRNA
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25
encoding both PR and PRLR is present in body cells,
but not cap cells, of the TEB in the mouse mammary gland, and is also heterogeneously distributed
within the ductal epithelium (Fig. 1(E) and (F)). The
distribution of these receptors appears to be remarkably similar to that of ER. Our recent work has
demonstrated that exogenous P stimulates ductal side
branching and associated TEB formation during allometric growth (58) while exogenous PRL stimulates
additional DNA synthesis in ductal MEC of peripubertal female mice (35). Mice lacking PR or PRLR
undergo full ductal elongation (85,86), indicating that
neither P nor PRL are critical for ductal elongation
per se. However, mice lacking PRLR have subtle defects in TEB development (87) while both PRKO and
PRLRKO mice have impaired ductal side branching
(87,88).
Members of the EGF family are recognized
mitogens for MEC (89) and function to influence
allometric ductal growth. Local release of EGF stimulates ductal development in ovariectomized mice
(90,91); as does TGF-α (92,93). Furthermore, suppression of systemic EGF levels by sialoadenectomy
leads to reduced ductal development (94). By contrast, high doses of EGF inhibit the growth of actively growing TEBs in the mammary glands of intact
mice (95). In addition to its presence in MEC, EGFR
is present in stromal cells, primarily as ErbB1 (96),
and is expressed in close proximity to TEB at a fivefold higher level than is found in more distal locations. Along these lines, local release of EGF stimulates stromal proliferation (90). The critical role for
the EGFR during ductal development is indicated by
the fact that mammary glands in EGFRKO mice do
not undergo ductal elongation due to a requirement
for stromal EGFR (46).
Hepatocyte growth factor/scatter factor (HGF/
SF) was originally identified as a mitogen for MEC
in conditioned medium from mammary fibroblasts.
Subsequently it has been identified as a stromaderived paracrine factor that regulates epithelial
growth and ductal morphogenesis. In addition to
its mitogenic effect, HGF has morphogenic effects
on MEC leading them to undergo tubulogenesis
and branching in collagen gels, similar to that observed during normal ductal elongation in vivo (97).
Locally synthesized transforming growth factor-β1
also likely regulates ductal development during allometric growth by either suppressing ductal elongation or regulating ductal spacing (98), or possibly by stimulating ductal branching morphogenesis
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in a fashion similar to that dictated by HGF (97).
All of these locally produced growth factors represent candidate effectors of hormone action, although many aspects of their regulation remain to be
elucidated.
Ruminants
The hormonal regulation of allometric growth
in ruminants appears to vary between species. In
heifers it is clear that prepubertal ovariectomy
abolishes subsequent mammary gland development
(70,99), despite seemingly insignificant changes in
serum E levels (99). In contrast, results from several studies (70,100) indicate that ovariectomy does
not negatively impact allometric mammary growth in
sheep. However, exogenous E stimulates MEC proliferation in both species (68,100) and restores ductal development in heifers following ovariectomy (70). Furthermore, it appears that ER localizes only to MEC
in the mammary gland of heifers (101); in contrast
to observations in rodents. Interestingly, a single report that examined the effect of P on MEC proliferation in prepubertal heifers indicated that it was
without effect, and suppressed the mitogenic effect
of E (68). Along with the critical role for E during
ductal development, at least in heifers, it is clear that
GH also directs allometric mammary growth in ruminants. Exogenous GH administered to intact peripubertal heifers and sheep stimulates mammary gland
development (99), but is ineffective in the absence of
E (99). While it was long believed that GH exerted
its mitogenic effect on ruminant epithelium via systemic IGF-I produced by the liver, it is now clear that,
as in rodents, there is likely a major role for stromaderived IGF-I. Indeed, in support of this proposal we
recorded increased expression of IGF-I mRNA in the
MFP of sheep (102) coincident with the prepubertal phase of allometric growth. Subsequent studies in
heifers have supported this conclusion (103). A correlation also exists between pubertal mammary gland
growth and pituitary PRL levels (18), although there
is no direct evidence that PRL regulates proliferation
or ductal morphogenesis during allometric growth
in ruminants. As in other species, the local synthesis of various growth factors including TGF-α (104),
various fibroblast growth factors (105); and HGF
(Hovey et al., unpublished observations) in the ruminant mammary gland may also influence allometric
mammary growth, although information concerning
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their hormonal regulation or precise contributions is
lacking.
Humans
The hormonal regulation of allometric growth
in humans is less clear. Elongation of the mammary ducts occurs with the onset of ovarian function at the start of puberty. Indeed, serum E levels
increase in girls during early puberty parallel to breast
development (106,107); while exogenous E stimulates breast development in hypogonadic girls (108).
Based on one study in girls, it is possible that E
also cooperates with LH and FSH to effect normal
breast development (109). While ERs are present in
MEC during this development (110), their precise
function is unclear, particularly given that stromal
ER probably does not exist in the developing human breast (111), and therefore does not fulfill the
same critical function identified in the mouse. TDLUs,
type 1 have a higher rate of proliferation than types 2
or 3, and consistently express both ER and PR in
a higher percentage of cells than type 2 or 3 lobules (110). The ER and PR colocalize in 96% of
PR-positive human luminal MEC (112). As in mice,
cells that express ER and PR are nonproliferative
(112,113), suggesting that E and/or P may stimulate
adjacent ER/PR negative cells to divide by a paracrine
mechanism (112). Despite these observations, it still
remains uncertain as to what specific effects E and/or
P impart during the proliferation and morphogenesis
of TDLUs, particularly type 1.
Information concerning the contribution of GH
to allometric mammary development in girls is lacking, although it is clear that serum GH levels increase
in girls during puberty (114). Furthermore, consistent with findings in other species, stromal cells in the
human breast express IGF-I mRNA, and its expression is increased in the vicinity of normal MEC (115).
Systemic IGF-I levels increase during puberty (116),
indicating that local IGF-I synthesis may also increase
within the breast during this period. Likewise, it is unclear whether PRL contributes to MEC proliferation
or morphogenesis during allometric growth. Serum
PRL levels increase during puberty in parallel with
serum E levels (106) and may therefore contribute to
aspects of TDLU development.
The limited availability of data in this area emphasizes that the endocrinological regulation of early
breast development is poorly understood. Given that
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this window is likely a major determinant in ultimate
breast cancer risk, many fundamental questions pertaining to this area require the prompt generation of
thorough answers.
Epithelial–Stromal Interactions
One of the major components of ductal elongation is the interaction between MEC and the adjacent
cells of the MFP in which it develops. In mice, the
MFP is primarily composed of white adipocytes (16)
that directly abut cap cells of the TEB. Fibroblastic
stromal cells flank the neck of the end bud, where
there is pronounced synthesis of extracellular matrix
molecules such as chondroitin sulfate (117), perhaps
in response to the newly differentiated myoepithelium and/or synthesis of the basal lamina. Interestingly, Daniel et al. (118) showed that TEB formation
depends on the interaction of MECs with adipocytes,
whereas the outgrowth of radial spikes can occur
by the interaction of MECs with collagen or the fibroblastic stroma. An interaction between MECs and
stromal cells is clearly evident where TEBs ramify
into the MFP. At these points stromal proliferation is
increased within 250 µm of the TEB and decreases
with distance. As indicated above, this proliferation
coincides with the induction of EGFR within the surrounding stroma.
The process of the epithelial–stromal interaction during hormone-induced ductal development in
humans and ruminants is less clear. Ductal elongation in both ruminants and humans is accompanied by a distinctive epithelial–stromal interaction
that results in the establishment of a loose proximal intralobular stroma that surrounds the epithelial
ductules, and a more dense, collagenous interlobular
stroma (16). Stromal cells in the human breast express
mRNA for PRLR and GH receptor (119), while in the
human (111) and heifer (101) they apparently do not
express ER or PR.
In contrast to observations in mice, treatment of
heifers with E results in the proliferation of stromal
cells after a round of epithelial proliferation (68). It
is clear from other more recent studies in ruminants
that hormones do influence the function of this stromal environment. For example, exogenous E downregulates levels of keratinocyte growth factor mRNA
in the ovine MFP, whereas it tends to upregulate
IGF-I and downregulate IGF-II mRNA expression.
Similarly, E increases the expression of IGF-I mRNA
27
in the bovine MFP. Furthermore, these responses can
be positively or negatively influenced by paracrine
signals from the adjacent epithelium (103,120).
TERTIARY BRANCHING AND
ALVEOLAR BUDDING
Histomorphology During the Estrous/
Menstrual Cycle
Rodents
The mouse has a 4–5-day estrous cycle that is divided into proestrus, estrus, metestrus, and diestrus.
Follicular growth is rapid during proestrus, and while
the thecal cells produce E, the follicle also produces
small amounts of P. The dominant follicle undergoes
ovulation 2–3 h after the start of estrus. Many corpora
lutea are present during metestrus, with one large corpus luteum, that secretes P for only a short period,
remaining at diestrus. The rat has either a 4- or 5-day
estrous cycle with hormonal changes similar to those
in the mouse, with an apparent extension of P secretion and diestrus during the 5-day cycle (121).
In the female mouse, ductal elongation and
branching continues after puberty until the mammary pad is filled by approximately 9–12 weeks of
age, depending on the strain (75,122). This expansion mainly occurs through the dichotomous branching of end buds, although lateral buds can also arise
through monopodial side branching (123). Between
the 12th and 16th week of age, limited growth and
regression occurs with each estrous cycle through increased branching and alveolar budding to establish
a finely branched ductal system with variable alveolar
development (75,122).
The glands of postpubertal female mice have
wide ducts and end buds of varying size embedded in
the MFP (75). After the onset of puberty, additional
development occurs in the form of tertiary branches
that extend from ductal side buds. Tertiary ducts are
thinner than primary or secondary ducts and are composed of a single layer of cuboidal MEC that surround
the lumen (62). Basal to these MEC is a monolayer of myoepithelial cells that forms a continuous
sheath around large ducts (124) but is discontinuous
around smaller tertiary ducts (2). A thin layer of dense
stroma consisting of connective tissue and fibroblasts surrounds the basement membrane of the ducts
(125).
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The readiness of the gland for pregnancyassociated development is indicated by the transitory appearance of alveolar buds during the estrous
cycle. These buds are composed of disarranged MEC
(Fig. 1(C)), while rudimentary alveoli are lined by a
single layer of epithelium (122) and secrete milk products in response to hormonal changes during the estrous cycle (122,126,127). Some strains of mice (RIII
and BALB/c) have few alveoli in their mature glands
(128,129), while other strains (C3H/He, C57/BL6,
FVB, and CD-1) have many alveoli (75,130). This variability in alveolar budding correlates with the length
of the estrous cycle luteal phase.
Cole (131) was the first to study morphological changes within the mammary glands of mice
during the estrous cycle. From these data it became evident that TEBs form during proestrus while
ductal extension and dilation occurs during estrus.
Decreases in duct width and the presence of TEBs occurs during metestrus while an open network of thin,
branched ducts is characteristic of diestrus. Andres
and Strange (132) found that the proliferation of
mouse MEC was highest during late proestrus and estrus and minimal during diestrus. In contrast, a recent
study by Fata et al. in mature (12- to 14-week-old)
mice demonstrated increased alveolar development
during diestrus without changes in ductal proliferation during the estrous cycle (130). Our observations
in pubertal BALB/c mice concur with those of both
Cole and Fata et al. in that maximal TEB development was observed in estrus (Fig. 1(B)) whereas alveolar budding was maximal at diestrus (Fig. 1(C)). High
levels of alveolar apoptosis appear to consistently occur during diestrus (130,132). However, it is still unclear whether it is a subset of MECs that terminally
differentiate and then undergo apoptosis during each
estrous cycle. Milk protein gene expression in young
nulliparous mice (4–6 weeks) is low in estrus, maximal
in metestrus (132), and absent at diestrus (126).
In Sprague-Dawley rats, the morphology of the
mammary gland changes from being predominantly
ductal at diestrus II to alveolar at metestrus, although
there is immense variation between animals at each
stage (127). The proliferation of luminal and myoepithelial cells in ducts and ductules in 25- to 60-dayold rats is lowest at early estrus and highest at late
estrus, while TEBs have two peaks of proliferation,
one at early estrus and one at metestrus (133). These
data concur with the findings of Sinha and Tucker
(134), who found that DNA content of rat mammary
glands was lowest in proestrus and maximal in estrus
and metestrus between 25 and 50 days of age. Two
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different studies in 4-day cycling adult rats found that
the overall proliferation of MECs is lowest during
proestrus and highest during metestrus and diestrus
I (127,135). The disparity between studies is hard to
reconcile, but may reflect different cycle lengths of the
rats used or their different ages and number of cycles
experienced (134).
Ruminants
The cow is the most widely studied ruminant in
the field of mammary biology, so discussions will be
restricted to this species. Unfortunately, very little
data exists regarding morphological changes in the
bovine mammary gland during the estrous cycle. At
puberty, heifers start their approximately 21-day estrous cycles, consisting of estrus at day 0 of the cycle,
metestrus from day 1 to 5, diestrus from day 6 to 17,
and proestrus from day 18 to estrus. Ductal development continues after the onset of puberty at an isometric rate, and full alveolar development does not
occur until pregnancy (136). In one study, changes
in mammary gland weight and content were quantified during the estrous cycle. The data showed increases in both DNA synthesis and the RNA/DNA
ratio during the estrous cycle that peaked at estrus,
and then declined during metestrus and diestrus (18).
These increases were accompanied by the formation
of large lobules with secretion into the alveolar lumen.
In contrast, during diestrus the lobules were smaller,
lacked secretion, and were lined by columnar alveolar
cells (18). Clearly there is much still to be learned concerning the hormonal regulation of ruminant mammary gland morphogenesis during the estrous cycle.
Humans
The human menstrual cycle typically lasts between 25 and 30 days. The cycle consists of two
halves; a follicular phase followed by the luteal phase.
Menstruation commences at day 0 and is followed by
a follicular phase with ovulation occurring in the middle (days 10–16) of the cycle. This is followed by the
luteal phase. As in the mouse, the ovary in humans
secretes E and a small amount of P during the follicular phase. In addition, the human is unique in that
the corpus luteum also secretes E in addition to P. This
section discusses the morphology of the human breast
and what is known of its development and hormonal
regulation during successive menstrual cycles.
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At the completion of pubertal growth, the breast
parenchyma is composed of a ductal system terminating in TDLUs, with the greatest proportion of
epithelium present in the outer third of the breast
plate (137). The degree of lobular complexity varies
greatly between women, between breasts, and within
a breast; full differentiation is a gradual process and
in some nulliparous women is never attained (12).
A TDLU, type 1, has been defined by Russo and
Russo (12) as a cluster of approximately 11 small
ductules or alveolar buds around a terminal duct
that is embedded in specialized intralobular stroma.
Type 2 and 3 TDLUs have approximately 47 and
80 ductules, respectively (12). Increases in lobular size
due to increasing numbers of ductules are accompanied by decreases in the size of the ductules (12).
Tertiary branching as described in mice is not recognized as such in the human. Instead, there is division of TEBs, either dichotomously or sympodially, to
give rise to segmental ducts and small subsegmental
ductules (12). It is not clear whether this side branching occurs during or after puberty, although it likely
occurs at all stages and varies between women.
With each menstrual cycle there is new budding until approximately age 35, when the gland
reaches a plateau in its growth (138). Morphological and histological modifications occur within the
breast during each cycle (139,140) in both the epithelium and stroma (139,141). The gland undergoes morphological changes during the menstrual
cycle (139,140). The follicular phase of the cycle
(days 3–15) is characterized by the presence of
small lobules with few acini, relatively few mitotic
figures, and dense cellular stroma (139,140). The
luteal phase (days 16–26) is characterized by welldeveloped lobules, acini with open lumens, prominent vacuolization of basal clear cells, and loose edematous stroma (139,140). Cell division is greatest in
the luteal phase of the cycle (140,142,143). However,
Vogel et al. (139) did not find this to be the case. From
Day 27 to menstruation the gland appeared to involute, with signs of epithelial degeneration, necrosis,
and a dense cellular stroma (139,140). This corresponds with the observations that during the luteal
phase, a peak in apoptosis closely follows (by approximately 3 days) a peak in mitosis (142,143). A drop
in proliferation and apoptosis with age (142) is consistent with the observation that the breast reaches a
plateau in its development by about age 35. Although
the proliferative status of the human breast during
successive menstrual cycles is known, it is unclear
whether this proliferation results in ductal growth,
29
alveolar budding, and/or TDLU maturation. Given
the distinction between morphological development
in the rodent and human mammary glands, it is clear
that such questions must be specifically addressed in
humans.
Hormonal Regulation During the
Estrous/Menstrual Cycle
Rodents
The ovarian hormones E and P fluctuate during
each estrous cycle and are critical for complete ductal development. There are also strain and species
differences in the profile of these hormones during
the estrous cycle. Interpretation of data from several studies indicates that the level of E peaks at
either proestrus (144) or estrus (130) in the mouse.
P levels peak at diestrus in the CD1 (144) and
C57/BL6 (130) strains of mice, although this timing is extremely strain-dependent (145). In the rat,
P is produced by the follicle during late proestrus
in response to luteinizing hormone (LH; 146), and
is secreted again at metestrus/diestrus by the corpus
luteum (121,147,148).
The combination of E, P, and either PRL
or GH is required for the mammary glands of
12-week-old ovariectomized mice to develop to an extent comparable to that in ovary-intact, 16 week-old
mice (75). Exogenous E stimulates proliferation in
less than 1% of ductal MECs in 10-week-old ovariectomized mice (149,150); rather, E-induced proliferation primarily occurs in cells (1.5–27%) within TEBs.
By contrast, P, in the presence of E, stimulates proliferation in approximately 4% of MECs in both ducts and
TEBs of 10-week-old ovariectomized mice, leading to
alveolar budding and ductal side branching (149,151),
as occurs in a cyclical manner during the estrous
cycle. This P-induced proliferation reflects the presence of E-inducible PR that occurs from approximately 7 weeks of age (40), and may also reflect
the fact that E sensitizes the gland to pituitary hormones (152) that may, in turn, interact with P to induce ductal side branching (35).
Tertiary branching in the mouse mammary gland
requires P. Although PRs in MECs are not essential for ductal elongation (153), they are required
for tertiary branching, alveolar budding and lobuloalveolar development (88). These findings concur
with our demonstration that exogenous P stimulates ductal branching in ovary-intact peripubertal
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Fig. 3. The effects of thyroid hormones and progesterone (P) on tertiary branching. Whole mounts of
mammary glands from mice are presented. (A) 3-month-old C3H mouse; (B) 3-month-old hypothyroid
C3H mouse, after treatment with propylthiouracil for 5 weeks; (C) 3-month-old hyperthyroid C3H
mouse, treated with thyroxine for 5 weeks; (D) 39-day-old BALB/c mouse (reproduced by permission
of the Society for Endocrinology; 58); (E) 39-day-old intact BALB/c mouse treated with P for 15 days
(reproduced by permission of the Society for Endocrinology; 58).
mice (Figs. 3(D) and (E); 58). Likewise, a correlation
exists between serum P levels and alveolar proliferation during the estrus cycle (130). Given that
PR-positive cells are generally nonproliferative (154),
it appears that P may initiate paracrine stimulation
as proposed by Brisken et al. (86), perhaps via pathways including the activation of Wnt-4 (155). However, in another study, Zeps et al. (156) proposed that
P directly stimulated proliferation of PR-positive cells
in the basal cell population, perhaps to form tertiary
branch points. Another important consideration during P-induced side branching is the ratio of the PRA and -B forms. Over-expression of PR-A results in
excessive side branching (157), while excess PR-B results in premature ductal growth arrest (158). Obviously there is a complexity of cell subpopulations and
cell–cell interactions contributing to P-induced tertiary ductal branching that remain to be understood.
In addition to the essential role for P, PRL is also
required for tertiary branching and alveolar budding
in the mouse mammary gland (87,159). Serum PRL
peaks at late proestrus in certain mouse strains (145)
and in rats (121,147), the latter also having an additional PRL peak during estrus (121,147). PRLR
in MECs is not essential for ductal side branching
or alveolar budding, but is absolutely required for
complete alveolar development (87). Therefore, the
action of PRL during tertiary branching and alveolar
budding is indirect and likely occurs through its action
on other tissues such as the ovary, or by its action on
mammary stromal cells. Along these lines we recently
described the spatio-temporal expression of all four
forms of the mouse PRLR in the MFP and the intact mammary gland during development (35). While
E and P clearly interact to stimulate ductal branching, our results also indicate an important interaction
between P and PRL. Whereas P or PRL alone fail
to stimulate epithelial proliferation in 10-week-old
ovariectomized mice, their effects markedly interact
to induce mitosis in approximately 35% of MECs
in the absence of E. These findings coincide with
the demonstration that PR and PRLR colocalize in
the mammary gland epithelium of sexually-mature
female mice, indicating the ability of these two
hormones to play a combined role during tertiary
branching.
Thyroid hormones also regulate tertiary branching and alveolar budding (Figs. 3(A), (B), and (C);
160). Hypothyroid mice do not develop tertiary
branches in their mammary glands (Figure 3(B))
while hyperthyroid mice display excessive branching
and alveolar budding (Figure 3(C); 160). Thyroid hormone levels generally remain constant, but the effects
of thyroid hormones appear to depend on their ratio
to PRL (161,162). It is possible that changes in the
ratio of PRL to thyroid hormones during the estrous
cycle may be involved in stimulating ductal branching
postpuberty. It is also conceivable that changes in the
ratio of either E or P to thyroid hormone during the
estrous cycle may also regulate tertiary branching.
Taken together, these data indicate that a complex combination of ovarian, pituitary, and thyroid
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hormones regulate tertiary branching and alveolar
budding in rodents. Although their individual effects
have been identified, the extent of their interactions,
particularly during the estrous cycle, has not been defined in any significant detail.
Ruminants
As in rodents and humans, the estrous cycle in
ruminants incorporates an estrogenic phase and a
luteal phase with concomitant changes in the levels
of E and P (163,164). Serum E peaks at estrus when
P levels are lowest, whereas serum P is highest during
diestrus. As in rodents, changes in the levels of PRL
and LH in the ruminant pituitary parallel the level
of P in the corpus luteum (18,163). It has been suggested that the levels of PRL and E are the principal
determinants of mammary growth during the estrous
cycle (18). Certainly in ewes the interaction of PRL
with E is essential for alveolar development (165,166),
while PRL is necessary for alveolar development in
cattle (167). However, in light of the importance of P
in the human and mouse mammary gland, this contention needs to be more clearly established. E and
P are known effectors of ovine and bovine mammary
ductal development (165,168), while EGF appears to
support maximal proliferation in transplanted bovine
mammary tissue exposed to E plus P (169).
From the limited information available it appears that parenchymal morphogenesis in ruminants
is somewhat analogous to that in humans. However,
much still remains unknown about the endocrine regulation of this development in either system, particularly during the estrous/menstrual cycle.
Humans
The follicular phase of the menstrual cycle is characterized by low levels of serum P and a peak in
serum E just prior to a peak in the level of LH.
The postovulatory luteal phase is characterized by an
extended elevation of serum P and a corresponding
peak in E. The level of ER in the breast declines between the follicular phase and the luteal phase (170),
whereas PR remains constant at a high level (171). On
average, serum E is higher in the luteal phase than in
the follicular phase. Since PR is inducible by very low
levels of E, it is not surprising that PR levels do not
change throughout the cycle (112).
Proliferative MECs in the breast are ER/PR
negative (110,112). This finding concurs with data
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from some studies with mice (154,155) but not others
(156). Proliferation of MEC in the breast correlates
with serum P levels during the menstrual cycle (172)
and is maximal during the luteal phase, peaking at
days 23–25 (140,143). This increase in proliferation
also coincides with a peak in E at days 22–24 (173). Despite the clear effect of P, this increased proliferation
appears to be due, at least in part, to E since tamoxifen can inhibit proliferation in normal breast tissue
during the luteal phase of the menstrual cycle (174).
Furthermore, human breast tissue transplanted into
athymic nude mice does not proliferate in response
to P (175,176). Despite this paradox, some evidence
points to an important role for P in MEC proliferation
in primates. Long-term treatment of surgically postmenopausal macaques with E plus P induces more
proliferation than E alone (177), while breast tissue
from women receiving E plus P during hormone replacement therapy is more proliferative than tissue
from women receiving E alone (178). Antiestrogenic
and antiproliferative effects of P on breast MECs
have also been documented (179), indicating that a
delicate balance of E and P directs normal breast
development.
While PRL has not been implicated as a major
factor in the growth and development of the human
mammary gland, its role has not been extensively
studied. A peak in serum PRL coincides with surges in
LH and E at the midpoint of the menstrual cycle (180),
similar to the situation in rodents (121,145,147). There
is also evidence that PRL or PL, in combination with
E, is mammogenic for human breast tissue (176,181).
Based on the potential role for PRL in breast
cancer (182), more studies are required to define the
role of PRL during normal breast development.
The specific regulation of normal postpubertal
mammary development by the stroma has not been
studied to a great extent in any species. However,
the expression of specific stroma-derived extracellular matrix components and their presence in the basement membrane markedly change during the menstrual cycle (141). These components include tenascin,
laminin, heparan sulphate proteoglycan, type IV collagen, type V collagen, chondroitin sulphate, and fibronectin, which may mediate hormonal effects on
the gland. By contrast, the levels of other extracellular matrix molecules do not change during the
menstrual cycle and are probably involved in structural support (141). Information implicating specific
growth factors in hormone-induced proliferation in
the normal human breast is scant. Based on studies in
other species, it is clear that factors such as IGF and
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EGF family members, HGF/SF, and various fibroblast
growth factors may fulfill such roles (183,184), although their precise contribution during these processes, particularly during the menstrual cycle, is
unclear.
Hovey, Trott, and Vonderhaar
Cruz, for graciously providing us with photomicrographs used in this article. R.C. Hovey is supported by
the US Army Medical Research and Materiel Command, DAMD 17-99-19311.
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CONCLUSION
The mammary gland relies upon a delicate balance of endocrine signals to achieve its ultimate morphology during stages from embryogenesis through
to sexual maturity. The hormones that are important
for ductal growth and morphogenesis change between
different stages and act together in a fine balance that
facilitates the correct morphological development of
the gland. Considerable evidence indicates that the
local synthesis of various autocrine and paracrine
growth factors mediates hormone action on the mammary gland. However, many of the specific regulatory
pathways underlying this mechanism of hormone action are still unknown.
Large species differences exist with respect to
both mammary gland morphology and the endocrine
regulation of ductal development. While the vast
majority of information concerning endocrine and
growth factor regulation of ductal morphogenesis
stems from studies in the mouse, much of this may
be irrelevant to studies in the human breast owing
to differences in the morphology of the mammary
gland between species. Whether alternative models
in other species would afford a more enlightening approach remains to be established. Overall, however,
the problem remains that little is known about development of the normal human mammary gland. Given
the current understanding that the proliferative state
of the breast influences its tumorigenic susceptibility,
it is of paramount importance that a more comprehensive understanding of its endocrinological regulation
be established. Without an adequate understanding
of the endocrine regulation underlying normal mammary gland development in humans, progression to
the cancerous state cannot be fully understood.
ACKNOWLEDGMENTS
We thank Dr Robin Humphreys, NIDDK, NIH,
Dr Jose Russo, Fox Chase Cancer Center, Dr Hugh
Dawkins, University of Western Australia, and
Dr Charles Daniel, University of California Santa
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