Seminars in Cell & Developmental Biology 23 (2012) 557–566

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Seminars in Cell & Developmental Biology

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Review

Growth and development of the mammary glands of livestock: A veritable

barnyard of opportunities
Ashley R. Rowson a , Kristy M. Daniels b , Steven E. Ellis c , Russell C. Hovey a,∗
a
Department of Animal Science, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
b
Department of Animal Sciences, The Ohio State University, Ohio Agricultural Research and Development Center, 1680 Madison Ave, Wooster, OH 44691, United States
c
Animal and Veterinary Sciences Department, Clemson University, 124 Poole Agricultural Center, Clemson University, Clemson, SC 29634-0311, United States

a r t i c l e i n f o a b s t r a c t

Article history: The mammary glands of all mammals are rich and diverse in their histomorphogenesis, developmental
Available online 3 April 2012 biology, genomics and metabolism. Domesticated livestock comprise a unique population for the analysis
of mammary gland and lactation biology, where much of what has been learned about these topics origi-
Keywords: nates from studies of these species. However, with the strong trend toward using rodents as flexible and
Mammary gland attractive models for normal mammary biology and cancer, there is a growing void of new information
Stroma
related to biology of the mammary glands in these relevant and informative domestic livestock. In turn,
Pigs
this trend threatens to reduce opportunities to either capitalize on an abundance of pre-existing data or
Cattle
Sheep
to apply this information to studies of lactation and cancer. Herein we review the unique and discerning
features of mammary gland development in several domestic livestock species including cows, sheep
and pigs and provide an overview of the factors regulating it. At the same time we discuss some of the
key considerations for studying these species, their limitations, and the associated opportunities. From
such an analysis it quickly becomes clear that much remains to be learned about the mammary glands of
domestic livestock, particularly given their many similarities to the human breast, the unique biological
mechanisms they employ, and the phenotypic variation they afford.
© 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
2. Fetal development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
2.1. Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
2.2. Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558
2.3. Sheep and goats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
3. Pre- and peri-pubertal development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
3.1. Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
3.2. Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
3.3. Sheep and goats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
4. Gestation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
4.1. Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
4.2. Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
4.3. Sheep and goats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
5. Involution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
5.1. Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
5.2. Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
5.3. Sheep and goats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
6. Discussion and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564

∗ Corresponding author.
E-mail address: rchovey@ucdavis.edu (R.C. Hovey).

1084-9521/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.semcdb.2012.03.018
558 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566
1. Introduction
The course of normal mammary gland development can be
broadly characterized as a progression of events that begins in
embryogenesis and ends with post-lactational involution. Ironi-
cally, many descriptions of these processes in the review literature
are often broad-stroked, and generally outline events and mecha-
nisms that have been resolved in a subset of rodents, namely rats
and mice. Yet, mice and rats are within but one order among >5600
mammals. Hence it seems far from appropriate to limit any sum-
mary of the processes underlying mammary development to these
few species.
Domesticated livestock, which for the purpose of this review
includes pigs, cattle, sheep and goats, are dual-toed ungulates in
the order Artiodactyla. While estimates vary, there are approxi-
mately 1.3 billion cattle, 1 billion each of sheep and pigs, and 700
million goats worldwide. The global economic value of these four
species combined is tremendous and ultimately hinges on their
mammary glands, either through their capacity to provide dairy
products, or because their growth and production of milk under-
lies successful animal production, welfare and survival. At the same
time, the widespread abundance of domestic livestock highlights
their potential as a model for diseases such as human breast cancer,
where recent advances on fronts such as whole genome sequencing
and proteomics stand to overcome many of the perceived obstacles
for utilizing these species.
In this review we seek to summarize the key processes of
mammary gland development in domesticated livestock. We focus
specifically on developmental changes and their regulation dur-
ing embryogenesis, prior to and during puberty, during gestation
and during post-lactational involution. As becomes quickly evident,
their mammary glands are notably different from those in rodents,
and in fact bear several similarities to the human breast. Many of
these comparisons are outlined in Table 1. We also consider the
pros and cons of studying the mammary glands of these species.
2. Fetal development
The mammary glands arise from the ectoderm during embry-
onic development to yield a rudimentary epithelial anlagen by
birth. These origins involve complex epithelial–mesenchymal
interactions and confer sexual dimorphism of the mammary glands,
which is species-dependent.
2.1. Pigs
Each gestation in pigs lasts approximately 115 d. Development
of the mammary glands of embryonic pigs was first described by
Schultze in 1892 and Rein in 1882, as last reviewed by Turner [1].
The future mammary glands begin to develop through cell migra-
tion along two ventral mammary lines that extend from the front
limb to the inguinal area [1]. This movement leads to ectodermal
thickening and emergence of the mammary hillock, which is appar-
ent around embryonic (e) d45. Each hillock transitions from a flat
disc to an elongated flask, ultimately giving rise to an epithelial
bud that is visible by e65 (Hovey et al., unpublished data). These
buds ascend at discrete sites destined to become the future nipple,
coincident with regression of the mammary line [1]. Nipple forma-
tion ensues by a coordinated process including proliferation of the
underlying mesenchyme, protrusion of the mammary bud [1] and
invagination into the dermis. It is worth noting that while there
has been discrepant use of the terms teat and nipple in the litera-
ture, our convention herein is that a teat has a single galactophore
(as found in mice, cows and sheep) whereas the nipple contains
multiple galactophores (as found in pigs, humans and most of the
order Carnivora).
By e85 the two mammary ducts extend from the nipple, rami-
fying into the underlying mesenchyme (Hovey et al., unpublished
data). The ducts continue to increase in complexity by e105, where
proliferation of epithelial cells at the distal ends permits their
branching into the surrounding mesenchyme. By the time of birth
each of the two primary sprouts has formed a 3–4 mm streak canal
with an underlying, widened lactiferous sinus [1]. Ducts emanat-
ing from the mammary bud are solid, uncanalized cords while the
distal ends of the mammary ducts have a wide lumen [1].
The ventral nipples along the “mammary chain” are usually
paired regularly and symmetrically, although it is also common to
find them irregularly unpaired, or offset [1]. Interestingly, arrange-
ment of the nipples in pigs is sexually dimorphic, where males tend
to have a more regular arrangement than females, leading Turner
to suggest that offset arrangement of the nipples may benefit piglet
access during nursing [1]. Furthermore, polythelia (supernumerary
nipples) on the hind legs is common in pigs, consistent with their
origins from the mammary line (Fig. 1).
The average number of nipples per pig is approximately 12,
but ranges from 8 to 18 [1]. The number of nipples is also breed-
dependent, ranging from an average of 12.5 in Durocs to almost 15
in Landrace, and correlates with litter size across breeds [2]. This
variation is particularly important for piglet growth and lactation
performance given that the average litter size for pigs has increased
notably in recent decades as the result of genetic selection. Interest-
ingly, several studies have identified a variety of genetic markers for
nipple number in pigs, although no single candidate has been iden-
tified [3]. Another determinant of nipple number in pigs is sex ratio
within a litter, where a greater number of male littermates leads to
females having fewer nipples [4]. This is likely due to intrauterine
exposure to androgens, as occurs in mice [4,122,123].
Inverted nipples, where the cratered nipple fails to protrude
from the body surface, occur in 7.6% to 30% of pigs, is heritable, and
varies in incidence across breeds [5]. This phenomenon is of sig-
nificant economic impact given the dysfunction of these teats and
an increased risk for mastitis [6]. Interestingly, inverted nipples in
pigs appear to arise from inadequate mesenchymal proliferation,
consistent with defective epithelial-stromal signaling in these ani-
mals [6]. Several candidate genes have been identified as pertaining
to this phenotype [7], where a polymorphism in the PTHR1 gene
correlated with its incidence [7]. A recent microarray analysis fur-
ther highlighted the influence of various growth factor-regulated
pathways in these processes [5].
2.2. Cattle
Turner [8] provides a thorough characterization of mammary
development in the embryo and fetus of cattle during an aver-
age gestation of approximately 280 d. At approximately e30 there
are noticeable parallel thickenings of the ectoderm in the inguinal
region along the ventral side that constitute the mammary bands.
Almost as soon as they are formed, the mammary bands become
known as mammary lines after e35 at a time when the mam-
mary anlagen are first discernable. Formation of the mammary
crests by e37 reflects the regional proliferation of cells along the
paired mammary lines, while neighboring cells along the line do
not proliferate. Two crests arise per mammary line, eventually giv-
ing rise to the four “quarters” of the udder. The mammary hillock
stage (∼e40) follows, during which time the proliferative epithe-
lial zones in each crest become rounded off and simultaneously
sink further into the mesenchyme. The last stage of embryonic
mammary development is the bud stage that occurs around e43.
At this stage sexual dimorphism becomes evident, where mam-
mary buds in females are smaller than in males. Mammary buds
 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566 559

Table 1
Comparative aspects of mammary gland development in mice, humans, pigs and ruminants.

Attribute Mice Humans Pigs Ruminants

Morphology Sparse ducts; TDLU TDLU TDLU

alveolar
Stromal histology [9] Adipose » connective Intralobular, Intralobular, Intralobular,
interlobular con- interlobular con- interlobular con-
nective » adipose nective » adipose nective » adipose
No. of ductules/TDLU (Types 1, 2, and 3, respectively) N/A 11, 47, 81 [114] 9, 24, 64 [22] Not defined
No. of galactophores 1 ∼8–15 2 1
Epithelial proliferation Concentrated in Concentrated in Concentrated in Peripheral Zones of
endbuds and endbuds or TDLU endbuds or TDLU TDLU [44]
alveoli
Response to E Endbud Epithelial [115] Endbuds, TDLU Epithelial [9,45]
Response to P Alveoli Negative, or No No Effect [22] No Effect [47,59]
Effect [116]
Mammary tumors Spontaneous, viral Spontaneous Rare, few cases Rare, few cases
origin [117] [118–120]
Tumor precursor AH, ADH, DCIS ADH, DCIS [121] Unknown Unknown
[121]
Tumor hormone dependence Rare [121] 50–60% [121] Unknown Unknown

Abbreviations: AH: alveolar hyperplasia; ADH: atypical ductal hyperplasia; DCIS: ductal carcinoma in situ; E: estrogen; P: progesterone; TDLU: terminal ductal lobular unit.

in females are also more ovoid in shape and are carried close
to the apex of the epidermis, giving the teats a slightly more
pointed shaped compared to teats in males. Mammary buds in
males are circular and are embedded by growth of the mes-
enchyme around them, eventually yielding teats that are broad
with flat ends. Unlike in mice where both a primary and a secondary
mammary mesenchyme can be recognized [9], only one type of
mesenchyme has been documented in the mammary glands of cat-
tle, although the precise nature of this dichotomy is unclear, as is its
speciation.

Fig. 1. Gross and histological aspects of mammary gland development in domestic livestock. (A) Ventral “mammary chain” of a nulliparous Yucatan pig depicting the frequent
asymmetric distribution of the nipples and glands in this species. (B) Sagittal bisection of one mammary gland quarter from an 8-week-old heifer showing the pigmented
parenchymal region within the surrounding mammary fat pad. (C and D) Hematoxylin stained mammary tissue from a prepubertal female ewe lamb (C) and a prepubertal
heifer (D). Note the abundance fibrous stroma and lobulated morphology. Scale bar = 50 ␮m.
560 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566
As the fetus develops the teat forms in each of the mammary
buds. Supernumerary teats arise from extra mammary buds and are
present in approximately 40–50% of females at birth. When present,
these extra teats are typically located behind the rear teats. Around
e65 the rapid proliferation of mesenchymal cells surrounding each
bud pushes it so that it protrudes above the existing plane of the
epithelium and skin. The previously round mammary buds then
begin to proliferate (ventrally to dorsally) in a lengthwise manner
to form a solid core of epithelial cells as primary sprouts, which are
first visible within the rudimentary teats around e80. If this sprout
fails to develop, the resultant gland lacks all subsequent glandular
development and will likely be a “blind” quarter. Secondary sprouts
begin to branch off primary sprouts around e90. Secondary sprouts
become the large milk-collecting ducts that empty into the gland
cistern.
Around e100 the primary sprouts undergo canalization that
yields the gland and teat cisterns. Each teat is canalized by one
primary sprout, where the tip of each teat invaginates from the
outside and forms the teat or “streak” canal that is lined by ker-
atinized epithelium. Further development of the gland and teat
cisterns mainly occurs through tissue remodeling and apoptosis,
where the gland cisterns develop first around e110, followed by the
teat cisterns around e130. The mammary rudiment of the bovine
female then undergoes its most rapid growth from the 7th month of
gestation [10], accompanied by expansion of the mammary fat pad
and development of the median suspensory ligament. With respect
to the nature and development of the mammary fat pad, Mayer and
Klein [11] state that in the neonatal calf, “the non-glandular struc-
tures are almost mature in form, with already established vascular
and lymphatic systems. The adipose and connective tissues are also
well organized. The early partitioning of the adipose tissue by the
connective tissue system is remarkable, and the connective septa
serve as paths for the future extension of the epithelial structures”.
It should be noted that while bulls have slower mammary
growth in utero, they retain a functional teat that is connected to
the parenchyma, as evidenced by the fact that these males can
be induced to lactate by administering exogenous female steroid
hormones in later life [12]. Another noteworthy aspect of mam-
mary growth in cattle is that females born to a twin male are
generally “freemartins” that lack full development of their repro-
ductive organs due to comingled fetal blood supplies and androgens
from the male calf that negatively impact development of the
female reproductive organs. Freemartin females also have sup-
pressed mammary development prior to 2 mo. of postnatal age
[13], which can be rescued, at least partially, by treatment with
exogenous ovarian steroids [14].
2.3. Sheep and goats
Information regarding fetal mammary development in sheep is
somewhat limited, and is scarce for goats. The mammary glands of
sheep and cattle generally undergo similar patterns of development
in utero [15,16]. A recent dissertation [17] provides an exhaustive
and well-illustrated analysis of fetal mammary development in
sheep, and supports the conclusion that mammary development
in fetal sheep follows essentially the same processes described for
cattle, with the exception that only two functional inguinal glands
form. The mammary glands of fetal male sheep grow at a constant
rate of 2.8 times body weight during a 150 d gestation while the
glands in females grow at 5 times body weight between e44 and
e70 [10]. Secondary ducts develop by e70, the teat cistern is evident,
and the parenchymal tissue is well developed. Mammary growth
then declines to 1.7 times the rate of body growth until birth [10].
Teat number in sheep can vary and has a high level of heritability,
where perhaps the best demonstration of this phenomenon came
from the personal flock of the infamous Alexander Graham Bell
who selected and bred ewes for teat number. These efforts real-
ized females with up to 8 teats, many of which were functional
[18]. A separate, more recent and intriguing line of investigation
indicates that modulating the nutrient intake by ewes can perma-
nently impact mammary development in their female offspring. For
example, Blair et al. [19] found that the level of maternal nutrition
(maintenance versus ad libitum diets) altered the fat yield and net
energy of milk from the female offspring. Meanwhile, Van der Lin-
den and colleagues [20] showed that the area of mammary ducts
in ewe lambs born to dams maintained on a high plane of nutrition
during pregnancy was reduced compared to that in ewe lambs born
to dams fed a maintenance diet. Given the widespread use of sheep
for studying maternal–fetal interactions, these findings reiterate
the potential to study maternal influences on mammary growth in
utero as well as postnatal mammary function.
3. Pre- and peri-pubertal development
Development of the mammary glands after birth coincides
with the transition from isometric growth to a state of allomet-
ric growth that facilitates rapid postnatal development. The onset
of this allometric growth may or may not be linked to ovar-
ian function. Likewise, the histomorphogenesis of this developing
parenchyma varies across species. Mayer and Klein [11] attributed
variation in parenchymal histomorphogenesis to the relative length
of the luteal phase during the estrous cycle. Such an association
should not be surprising given the well-established influence of
progesterone on alveolar growth [21]. Consequently, only ductal
morphogenesis is generally evident prior to the onset of pregnancy
in species such as rats and mice where the follicular phase pre-
dominates. In contrast, the mammary glands of species that have
a long luteal phase may display lobuloalveolar development along
the lines of that seen during pregnancy [11].
3.1. Pigs
Prior to puberty the mammary glands of pigs undergo grad-
ual ductal elongation and canalization [1]. Primary and secondary
ducts widen, and arborization of the ductal network occurs such
that secondary ducts branch into lateral ducts which further ter-
minate as ductules [1]. A similar complex branching pattern has
been described for the human mammary glands that can be equally
classified at the histomorphological level in both species as ter-
minal ductal lobular unit (TDLU) structures [22,23]. The onset of
puberty in female pigs occurs around 5–6 months of age. To our
knowledge there is no specific data regarding the rate of mam-
mary growth in female pigs prior to puberty, or its precise timing
relative to the onset of ovarian function. However, it is clear that
the mammary glands of pigs transition in complexity from TDLU-1
to -2 during this period due to the simultaneous proliferation of
lobular buds and ductal elongation driven by terminal end buds
[22]. By all accounts these terminal end buds are synonymous with
those found in mice [24], as evidenced by their high incidence of
epithelial proliferation, bulbous shape and undifferentiated cytol-
ogy. Information regarding the regulation of these structures and
all aspects of mammary growth in nulliparous female pigs and the
mechanisms underlying these phenotypes is scant. The incidence
of terminal buds in the mammary glands of ovariectomized female
pigs was markedly enhanced by exogenous estrogen or estrogen
plus progesterone [22]. By contrast, exogenous progesterone sup-
pressed the proliferation of the mammary epithelium [22], as also
noted in rats [25] and humans [26]. Whereas exogenous prolactin
alone failed to stimulate growth in ovariectomized female pigs [22],
it induced precocious mammary growth and lactogenesis when
 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566
administered to ovary-intact females [27], thereby emphasizing the
requirement for prolactin to act in concert with estrogen [22].
The nutritional modulation of mammary growth in prepubertal
female pigs has been a focus of recent investigations by Farmer et al.
Feed restriction early in life decreased mammary growth in female
pigs [28] whereas altering the profile of fat intake had little effect
[29]. Modifying nutrient intake by female pigs during prepuberty
only had subtle effects on their mammary growth by the end of
gestation, or on their subsequent lactation performance [30].
3.2. Cattle
The mammary parenchyma in newborn female calves exists
in negligible quantities and the mammary fat pad is barely pal-
pable, where the udder lies in close proximity to the body wall.
Meyer et al. [31,32] comment that the parenchyma at this stage
only comprises a threadlike mass that extends dorsally above each
teat. By 9 wk of age (∼80 kg BW) it is not uncommon to palpate
an almond-sized mass of parenchyma lying at the base of each
teat that weighs ∼2.5 g, in addition to one grapefruit-sized mam-
mary fat pad that weighs in excess of 80 g [33]. By this time the
parenchyma consists of a gland cistern and a ductal system lined
with double-layered epithelium that is accompanied by termi-
nal alveolar structures [11]. During normal prepubertal mammary
growth, mammary epithelial cells in heifers advance into the mam-
mary fat pad via ductal branching and elongation. In fact, this
growth corresponds to a 60-fold increase in the mass of the mam-
mary parenchyma between birth and 3 mo of age (Akers et al.,
unpublished data).
Sinha and Tucker indicated that positive allometric mammary
growth in heifers commenced at 2–3 mo. of age; the rate of increase
in mammary DNA content was then 3.5 times that for metabolic
liveweight until 9 mo. of age [34]. More recently, Meyer et al. [31]
suggested that allometric growth of the mammary glands in heifers
commenced from birth. It is possible that genetic or management
factors contribute to this different timing for the onset of allo-
metric growth in these studies. Puberty is realized at ∼280 kg in
large-breed dairy heifers such as Holsteins, which generally occurs
around 9 to 11 mo. of age. Thereafter mammary growth slows so
that the amount of DNA in the mammary glands of 12- and 16-
mo. old heifers is not different [34]. Quantitatively, more mammary
growth and development occurs during the pubertal period com-
pared to the prepubertal period, coincident with growth of the
parenchyma toward the bounds of the mammary fat pad. Ducts
continue to lengthen and branch in a ventral-to-dorsal fashion. It is
not uncommon to see secretion in ductal lumens at the histological
level, although alveoli are absent.
Similar to rats, parenchymal development in cattle increases by
about 8% per estrous cycle [35]. During each 21 d estrous cycle the
DNA content of the heifer mammary gland is greatest at estrus rel-
ative to other stages [34]. This cyclical variation is accompanied by
increased secretory activity at estrus when epithelial cells assume
a cuboidal appearance. Collagen content, as measured by hydrox-
yproline, is also elevated in the heifer mammary gland at estrus
[34]. Most dairy heifers become pregnant for the first time when
they are approximately 15 mo. so they can calve around 24 mo
of age. Therefore, most nulliparous heifers experience about 5–9
estrous cycles before becoming pregnant.
It is worth noting that ovariectomy of prepubertal dairy heifers
curtails further mammary development, where the earlier in life
the procedure is performed, the more drastic the result [36,37].
The period of positive allometric mammary growth in prepubertal
and pubertal heifers also corresponds to increased pituitary pro-
lactin levels [34]. In contrast, other studies that have investigated
the influence of liveweight gain on mammary gland development
of prepubertal heifers [38] and lambs [39] have demonstrated a
561
strong negative correlation between serum prolactin levels and
parenchymal growth during this period.
A considerable body of evidence has accumulated over the
past 50 years to suggest that overnutrition of heifers reduces
mammary growth and subsequent lactation performance. Using
identical twins, Swanson [40] first demonstrated that the reduced
lactational performance of heifers fed a high level of concentrate
was the result of impaired mammogenesis. Numerous reports
subsequently indicated that accelerated liveweight gain during
prepuberty and puberty had a detrimental effect on the subsequent
milk yield from dairy heifers [41], which was essentially irreversible
and remained in subsequent lactations [40]. The milk production
potential of ewes [42] and beef heifers [43] was similarly compro-
mised by high rates of liveweight gain. This phenomenon led to
many years of research on this topic, often with conflicting results.
For the purposes of this discourse, suffice it to say, the composition
and size of the mammary fat pad can be affected by level of nutri-
ent intake, whereas the composition of mammary parenchyma is
apparently unaffected [32,33].
More recent efforts have focused on describing histomorpho-
genesis of the developing mammary glands in heifers, where a
comprehensive study by Capuco et al. [44] is noteworthy. There
are several points to be made about this histomorphogenesis. The
stroma within the mammary glands of cattle is more fibrous and
contains less adipose tissue than the mammary fat pad of rodents
[9]. This fibrous stroma is also present in both inter- and intra-
lobular compartments [9]. Furthermore, the parenchyma of heifers
develops as a ductal-lobular network with peripheral zones of
epithelial proliferation [44]. Notably, end bud structures are not
present in the mammary parenchyma from either heifers or ewe
lambs [44,45].
Although ovariectomy prior to puberty abrogates mammary
development in heifers [46], a role for estrogen in this effect
is somewhat unclear given that ovariectomy only reduced the
serum estrogen concentration by 0.1 pg/ml. Indeed, short-term
administration of supraphysiological doses of estrogen to heifers
increased DNA synthesis by the mammary epithelium, which was
followed by a phase of stromal DNA synthesis [47]. It is also
well established that heifers and ewes grazing pastures with a
high phytoestrogen content frequently demonstrate precocious
mammary development [48]. Others have shown that estrogen,
in combination with progesterone, can facilitate normal growth
in ovariectomized mature heifers [49], where the combination of
these two steroids is essential to adequately develop the mammary
gland during hormone-induced lactation [50]. Conversely, admin-
istering progesterone alone to prepubertal heifers for 4 days did
not stimulate DNA synthesis within mammary tissue [47]. Admin-
istration of growth hormone to heifers [46] stimulates substantial
parenchymal development, although the resultant morphogene-
sis has not been described. Meanwhile, specific binding of IGF-I to
mammary parenchymal tissue of heifers is unaltered by exogenous
growth hormone, and is slightly increased by ovariectomy [46].
Administering IGF-I via the teat of heifers also leads to increased
parenchymal growth [51], whereas intramammary administration
of leptin abrogates epithelial proliferation [52].
3.3. Sheep and goats
A period of rapid mammary parenchymal growth was recorded
in ewe lambs between 8 and 12 [53], and 12 and 16 wk [54] of
age, well before the onset of puberty between approximately 6
and 8 mo. of age. Similarly, Johnsson et al. [55] recorded positive
allometric mammary growth in ewe lambs between 4 and 20 wk.
Thereafter, the mammary parenchyma grows toward the bounds
of the mammary fat pad [56] with a histomorphogenesis similar to
that described above for cattle.
562 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566
Perhaps one of the most interesting aspects of mammary growth
prior to puberty in sheep and goats is the potential for its dif-
ferential endocrine regulation. Wallace [53] first suggested that
ovariectomy of prepubertal lambs failed to impair mammary devel-
opment, although that report contained limited data from few
animals. Those observations were later confirmed by Ellis et al.
[45] who showed that the ovaries were not required for allomet-
ric growth of the parenchyma through at least the first 13 wk of
age. The mechanisms that underlie this intriguing and seemingly
unique independence from ovarian secretions remain unresolved.
In contrast, mammary development in goats during a similar pre-
pubertal interval apparently does require ovarian activity [57].
Although these studies in sheep and goats are not directly compara-
ble because of differences in the ages at ovariectomy and intervals
to necropsy, Dessuage and colleagues [57] clearly showed that pre-
pubertal mammary development, epithelial cell proliferation, and
gene expression in goats were negatively impacted by ovariectomy,
similar to reports in cattle.
Conversely, short-term administration of supraphysiological
doses of estrogen to prepubertal ewe lambs [45] increases DNA
synthesis by the mammary epithelium. Regarding a role for pro-
lactin during this window, administration of bromocriptine to
prepubertal ewe lambs had no effect on their mammary develop-
ment [58], whereas administering growth hormone to prepubertal
and pubertal ewe lambs [58] stimulated substantial parenchymal
growth. Meanwhile, treatment of post-pubertal ewes with proges-
terone suppressed epithelial proliferation [59]. The realization of
complete lobuloalveolar development in ewes ultimately requires
estrogen + progesterone in the presence of endogenous prolactin
[59].
Other studies indicate that nutritional modulation during the
prepubertal period can affect mammary development and subse-
quent milk production [60]. Restricting the feed intake of goats
affected mammary development [61], although the effects seemed
to occur primarily in the mammary fat pad, not the parenchyma.
Along similar lines, feeding high levels of protected polyunsat-
urated fats to prepubertal ewe lambs increased the amount of
mammary parenchymal tissue [62].
A limited mixture of data exists regarding the local growth reg-
ulatory mechanisms at play in the developing mammary glands
of sheep and goats. Expression of IGF-I receptor mRNA localizes
to epithelial cells in the fetal and prepubertal ovine mammary
glands, where its expression is greatest at 4 wk of postnatal age
[63]. The abundance of IGF-II mRNA in fetal ovine mammary tissue
increases with gestational age and is reduced in 4-wk postnatal
and adult mammary tissue [63]. In keeping with this timing, we
demonstrated that local IGF-I and -II expression in the mammary
glands of ewe lambs is elevated during the peripubertal period, and
is modulated by epithelial–stromal interactions [64]. As might be
expected, other growth-regulatory molecules such as keratinocyte
growth factor [65] and amphiregulin [66] are also expressed in the
developing mammary glands of sheep.
4. Gestation
The onset of pregnancy initiates considerable expansion of the
mammary parenchyma through alveologenesis in preparation for
the ensuing lactation. While the fundamental principles of this
alveolar expansion are consistent, there are notable differences in
this process and its regulation depending on the species in question.
4.1. Pigs
The mammary parenchyma of pigs undergoes considerable
development during gestation as it transitions from a TDLU-2
histomorphogenesis to the more-lobulated TDLU-3 morphotype
and its associated alveologenesis. The majority of mammary
growth in pigs occurs during the last trimester of a 115d gesta-
tion, as evidenced by a three-fold increase in the DNA content
of the mammary glands between d75 and d90 [67]. Histological
analysis at d90 indicates that the majority of alveoli are arranged
as a single layer of epithelial cells with large, irregular nuclei,
short microvilli at the apical surface with some basally located
lipid droplets [68]. Further differentiation of the epithelium by
d105 leads to a cuboidal epithelial morphology, regular-shaped
nuclei, increased size of lipid droplets and translocation of the Golgi
apparatus from a para- to supranuclear location [68]. By d112 the
cytoplasmic compartment is largely dominated by lipid droplets,
the nucleus has condensed, protein granules are apparent in the
Golgi apparatus and there is elongation of the microvilli [68]. By
d112 of gestation there is a dramatic increase in epithelial RNA
content [67] whereas lipid droplets are retained toward the basal
surface [68].
Using ovariectomized females we recently identified that the
transition from a TDLU-2 to TDLU-3 histomorphogenesis, as occurs
during gestation, requires the coordinated effects of estrogen and
prolactin, which is partly mitigated by progesterone [22]. This com-
plex interaction likely reflects changes including estrogen-induced
prolactin receptor expression in the mammary epithelium, thereby
increasing tissue sensitivity to its ligand [69]. These data support
previous findings that estrogen alone promoted modest devel-
opment of the parenchyma whereas progesterone either had no
effect [70] or negatively influenced epithelial growth [71]. Like
for most species, prolactin has a considerable effect on mam-
mary growth during gestation in pigs as revealed from studies by
Farmer et al. [72,73]. By administering bromocriptine at different
stages of gestation to suppress the release of pituitary prolactin,
these authors established that mammary growth in pregnant pigs
requires prolactin during a specific period from d90 to d109 of ges-
tation [72]. Like prolactin, ovary-derived relaxin also synergizes
with estrogen to markedly enhance lobulo-alveolar development
[70]. Interestingly, co-administering progesterone with relaxin
disrupted parenchymal histomorphogenesis while degrading the
collagen matrix surrounding the epithelium [70].
4.2. Cattle
Epithelial growth within the mammary glands of ruminants
such as cattle is allometric during pregnancy, and in heifers equates
to a cell doubling time of approximately 87d [74]. During gesta-
tion the mammary parenchyma of ruminants gradually replaces
the interspersed adipose tissue as it undergoes extensive alveo-
lar development [35], concurrent with extensive tissue remodeling
and condensation of the stromal connective tissue to narrow
bands. The total amount of mammary parenchyma inflects around
110–140d in cows when there is noticeable formation of alveoli-
filled lobules. Development of the ruminant mammary gland differs
from that for rodents in that the ruminant gland is almost com-
pletely developed at term, such that under normal conditions total
mammary DNA content of each gland does not change after partu-
rition in heifers [75], goats [76], or sheep [54]
Development of the mammary glands of ruminants is influ-
enced by a variety of maternal and fetal factors. In most species
there is a mid-gestational increase in the level of systemic placen-
tal lactogen that may suppress serum prolactin levels. Mammary
development occurring after this hormonal switching can be main-
tained by placental lactogen in these species, and to a lesser extent
by prolactin [77]. Administration of recombinant placental lacto-
gen to steroid-treated heifers stimulated substantial increases in
mammary development [77], where Collier et al. suggested that
placental lactogen stimulates mammary growth via an indirect
 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566
mechanism, based on their finding that it did not stimulate the
growth of cultured bovine mammary epithelial cells [77].
A lesser role for prolactin during mammary growth in late preg-
nancy is indicated by the fact that the mammary glands of goats [78]
and sheep [59] undergo extensive development in the absence of
prolactin during this period, although a periparturient surge of pro-
lactin is required for full mammary differentiation and optimal milk
synthesis [79]. Prolactin may play a more important role during
this latter phase of gestational mammogenesis in those species that
apparently lack a placental lactogen, except in pigs, where estro-
gens may be of greater importance [80]. Administration of growth
hormone to prepartum heifers [81] increases mammary develop-
ment, where in the mammary glands of cattle [82] there is an overall
decrease in the number of IGF-I binding sites across pregnancy and
into lactation along with a concomitant rise in IGF-I binding affinity.
4.3. Sheep and goats
Following allometric growth during gestation the total amount
of mammary parenchyma inflects around 70–80d in goats and
around 80–115d in sheep as alveoli become engorged. The high-
est percentage of epithelial tissue and proliferating epithelial cells
in the mammary glands of ewes is around d115 of gestation [83].
This period is also associated with an increase in epithelial cell size,
a general appearance of secretory activity within the mammary
gland, and an elevation in lactose synthesis and the ratio of RNA to
DNA [75].
Sheep have long served as a useful species for gestational mam-
mary development, though often in the context of biomedical
models for placental insufficiency or maternal undernutrition (see
[84]). Davis et al. [85] made the interesting observation that udder
growth in hemimastectomized ewes remained at 50% of controls
until d144 of pregnancy, after which the single gland underwent
compensatory growth to contain 70% of the DNA in control udders
at term. Although the mechanisms that promote this compensatory
growth are unknown, such a response illustrates the substan-
tial growth potential of the mammary glands during pregnancy.
Additional evidence for placental regulation of mammogenesis is
provided by the fact that a positive relationship exists between the
number of offspring and milk yield in goats and sheep [86], where
plasma placental lactogen levels in sheep begin to increase around
d50 of gestation and reach maximum levels just prior to parturition.
Recent studies have focused on how maternal nutrition impacts
mammary development and its productivity. For example, data
from Vonhamme et al. [87] demonstrate that supplemental
selenium can enhance angiogenesis and colostrum quality in prim-
iparous ewes, though earlier reports failed to show a pronounced
effect of selenium on mammary development [88]. Such efforts
illustrate both the need for additional study and the manifest
potential for discovering basic biological mechanisms that can be
translated to agricultural settings.
5. Involution
Following the cessation of milk removal the mammary glands
undergo dramatic regression, although this is somewhat species-
specific. Regardless, involution clearly is a period for tissue
remodeling and epithelial regression in all species, where the
extent to which this occurs differs significantly across species.
5.1. Pigs
Termination of milk removal from the sow upon weaning dur-
ing the third or fourth week of lactation leads to rapid regression
of the mammary glands. Turner [1] observed enlargement of the
mammary glands during the first 24 to 30 h post-weaning, while
563
studies by Cross et al. [89] indicated that engorgement was com-
plete within 24 h post-weaning. By 48 h post-weaning, little fluid
is evident in the alveolar lumens, suggesting that reabsorption
has occurred [89]. This suggestion is supported by pronounced
decreases in mammary gland mass and total DNA content such that
there is a 25% loss in total parenchymal area and a parallel decline
in wet weight [90].
Given the significant loss in total DNA content during the first 2d
post-weaning, it is likely that early involution in the sow is accom-
panied by significant apoptosis [90]. A similar loss in cell number
by apoptosis was been noted for mice [91] and to a lesser extent
in ruminants [92–94]. While few alveolar structures remain in the
sow mammary gland 4d after weaning [89], it remains possible
to obtain secretion by administering oxytocin [95]. The composi-
tion of the secretion is substantially different from normal milk
due to decreased lactose and increased sodium, consistent with
the break-down of epithelial tight junctions [95]. Involution of the
sow mammary glands is complete by d8, as evidenced by declining
parenchymal DNA content through d7 [90] and complete regres-
sion of alveoli by d8 [89].
5.2. Cattle
Without regular milk removal, the mammary gland regresses
back to a more undifferentiated state, which is referred to as “the
dry period” for dairy cows. The initial active phase of involution
takes about 30d, the following steady-state varies in length, and the
subsequent renewal period takes about 30d. These intervals form
the basis for the 60d dry period that is commonplace for most dairy
cattle. In species that do not experience concurrent pregnancy dur-
ing involution (most mammals except dairy cows), the involution
process leaves the gland having an appearance like that in a vir-
gin animal, whereby most alveoli regress. Because dairy cattle are
usually also pregnant during involution, the mammary glands do
not completely regress back to the virginal state, and many alve-
oli remain [96]. For a more thorough analysis of changes in the
bovine mammary gland during involution the reader is directed to
a comprehensive review by Capuco and Akers [97].
5.3. Sheep and goats
The available data on involution in sheep and goats [98,99]
demonstrate that over a brief interval after weaning or milk sta-
sis, the alveolar architecture degrades, the incidence of apoptotic
figures or TUNEL staining becomes more frequent, and expression
of pro-apoptotic genes is up-regulated in a manner consistent with
that recorded for other livestock species [100,101].
6. Discussion and future perspectives
From the above summary it is clear that while fundamental
aspects of mammary growth have been defined for pigs, cattle,
sheep and goats, there is a paucity of information regarding the
regulation of mammary development in these species, from the
histological to the molecular level. Much of this situation reflects
the fact that rodents have become the principle model for study-
ing mammary gland biology. To be sure, rodents offer several
important and specific benefits, such as low cost, short genera-
tion interval, the need for few and compact specialized facilities, an
abundance of reagents and the ability to manipulate their genetics.
As a result, numerous genetically identical mouse strains have been
developed, permitting evaluation of the functional significance
of specific genes under controlled conditions [102]. Transplanta-
tion studies using inbred and specialized immunodeficient mouse
strains have been particularly valuable for understanding mam-
mary development and tumorigenesis [103]. Nevertheless, there
564 A.R. Rowson et al. / Seminars in Cell & Developmental Biology 23 (2012) 557–566
are several important limitations associated with these models.
Importantly, the mouse mammary glands have limited translation
to other species given that the histoarchitecture and hormonal
control of the rodent mammary glands is often significantly dif-
ferent from that in other mammals. Furthermore, the small size of
rodents often precludes the collection of adequate sample volumes
or repeated sampling of the mammary glands, making studies of
milk composition and treatment responses over time challenging.
Two examples highlight distinct differences between the mam-
mary glands of rodents and domestic livestock. First, similar to
transplanted human cells, bovine cells do not outgrow in the mam-
mary fat pads of athymic nude mice, but instead form hollow
spheroid structures ensheathed by connective tissue [103]. It is
likely that the mouse mammary fat pad does not provide a stromal
environment suitable for the outgrowth of either human or bovine
mammary epithelium [9]. Indeed, Sheffield [103] reported that
the composition of the extracellular matrix surrounding spheroids
of heterografted bovine epithelium differed to that in normal
bovine mammary tissue. Second, progesterone appears to have
divergent effects in mice and other species having a prolonged
luteal phase, including domestic livestock. Progesterone admin-
istered to mice stimulates epithelial proliferation and ductal side
branching [95,104] whereas it has limited effects on the mammary
epithelium in ruminants, humans, and rats having a more complex
parenchymal morphogenesis. These differential responses suggest
that species differences in progesterone responsiveness may dic-
tate mammary gland morphology.
Looking to the future, domestic livestock offer several opportu-
nities to overcome certain biological limitations associated with
studying the mammary glands of rodents. Large animal models
often provide an exact or approximate model of the human con-
dition, such as bovine models of reproductive aging [105], sheep
models of fetal and postnatal development [102] and pig models
of spontaneous melanoma [106]. The similar histomorphogenesis
of the mammary glands in pigs and the human breast [22] makes
it a promising model for human breast development and disease.
In fact, multiple Nobel prize winning efforts have involved large
animal models [107], suggesting these models may offer novel
insights and opportunities for mammary biologists. Advances in
molecular techniques have facilitated complete, nearly complete
or partial genomic sequencing of the cow [108], pig [109] and
sheep [110], respectively. New technologies such as RNA-Seq and
proteomics can be used without the need for full annotation of a
genome or proteome. Analysis of large animal genomes will sig-
nificantly enhance the utility of these models for agricultural and
biomedical research [111]. For example, functional and compar-
ative genomic studies have determined the genetic diversity of
sheep breeds [112], uncovered genes of potential importance to
bovine lactation [113], and have revealed that features of the bovine
and porcine genomes are closer to the human genome than is the
murine genome [108,109]. Other techniques such as MG biopsy
allow for the repeated sampling of a given individual mammary
gland over a long period while providing substantial amounts of tis-
sue. Sheep and swine are particularly attractive models given their
similarities in body size and physiology with the human [102,106],
offering the opportunity to study complex biological interactions
on a whole animal level.
As for rodents, there are disadvantages associated with the use
and adoption of large animal models for studying mammary gland
biology. Perhaps the most obvious is the greater costs and logis-
tical challenges associated with the purchase and maintenance of
these species and the limited number of researchers with access to
appropriate training and facilities. Furthermore, the foundation of
large animal research itself continues to be challenged by a decline
in both the number of researchers trained in animal science and the
funding available for such research [107]. Regardless, the success
and translation potential of research depends upon appropriate
model selection, with large animal models promising novel oppor-
tunities to enhance not only agricultural productivity, but also our
understanding of mammary gland biology and human breast can-
cer.
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