(2012), 6:3, pp 355–368 &The Animal Consortium 2011
The evolution of milk secretion and its ancient origins
O. T. Oftedal
Smithsonian Environmental Research Center, Smithsonian Institution, Edgewater, MD 21037, USA
(Received 8 August 2011; Accepted 8 September 2011; First published online 14 October 2011)
Lactation represents an important element of the life history strategies of all mammals, whether monotreme, marsupial, or
eutherian. Milk originated as a glandular skin secretion in synapsids (the lineage ancestral to mammals), perhaps as early as the
Pennsylvanian period, that is, approximately 310 million years ago (mya). Early synapsids laid eggs with parchment-like shells
intolerant of desiccation and apparently dependent on glandular skin secretions for moisture. Mammary glands probably evolved
from apocrine-like glands that combined multiple modes of secretion and developed in association with hair follicles. Comparative
analyses of the evolutionary origin of milk constituents support a scenario in which these secretions evolved into a nutrient-rich
milk long before mammals arose. A variety of antimicrobial and secretory constituents were co-opted into novel roles related to
nutrition of the young. Secretory calcium-binding phosphoproteins may originally have had a role in calcium delivery to eggs;
however, by evolving into large, complex casein micelles, they took on an important role in transport of amino acids, calcium
and phosphorus. Several proteins involved in immunity, including an ancestral butyrophilin and xanthine oxidoreductase, were
incorporated into a novel membrane-bound lipid droplet (the milk fat globule) that became a primary mode of energy transfer.
An ancestral c-lysozyme lost its lytic functions in favor of a role as a-lactalbumin, which modiﬁes a galactosyltransferase to
recognize glucose as an acceptor, leading to the synthesis of novel milk sugars, of which free oligosaccharides may have predated
free lactose. An ancestral lipocalin and an ancestral whey acidic protein four-disulphide core protein apparently lost their original
transport and antimicrobial functions when they became the whey proteins b-lactoglobulin and whey acidic protein, which
with a-lactalbumin provide limiting sulfur amino acids to the young. By the late Triassic period (ca 210 mya), mammaliaforms
(mammalian ancestors) were endothermic (requiring ﬂuid to replace incubatory water losses of eggs), very small in size (making
large eggs impossible), and had rapid growth and limited tooth replacement (indicating delayed onset of feeding and reliance
on milk). Thus, milk had already supplanted egg yolk as the primary nutrient source, and by the Jurassic period (ca 170mya)
vitellogenin genes were being lost. All primary milk constituents evolved before the appearance of mammals, and some
constituents may have origins that predate the split of the synapsids from sauropsids (the lineage leading to ‘reptiles’ and birds).
Thus, the modern dairy industry is built upon a very old foundation, the cornerstones of which were laid even before dinosaurs
ruled the earth in the Jurassic and Cretaceous periods.
Keywords: evolution, lactation, milk composition, casein, milk fat globule
Lactation has an evolutionary origin and biological context
within which its secretory processes and milk constituents
evolved. This review provides an evolutionary scenario
within which the speciﬁc functions of milk constituents can
be investigated. It is hoped that this review will stimulate
increased attention to the evolution of lactation and milk
constituents in a large variety of disciplines, including mole-
cular biology, evo-devo, comparative genetics, protein struc-
tural studies, dairy chemistry, paleobiology, phylogenetic
systematics, mammary gland biology, reproductive evolution,
and comparative nutrition. The goal is an in-depth under-
standing of how the remarkable process of lactation came
Introduction: the importance of milk to mammals
Milk secretion is a characteristic feature of all mammalian
species, large or small, social or solitary, arctic or tropical.
The famous taxonomist Carolus Linnaeus selected the name
Mammalia in 1758 in uniting terrestrial ‘quadrupeds’ with
dolphins and whales to reﬂect the fact that females of both
groups bear mammae, or mammary glands (Gregory, 1910).
Lactation is unique to mammals. No other extant organism
produces copious glandular skin secretions to feed its young,
although the larvae of some ﬁsh ingest modest amounts of
nutritive mucus from surface mucous glands (Buckley
2010); terrestrial-breeding frogs provide water, antimicrobial
compounds, and perhaps some nutrients to their eggs via
granular and/or mucous gland secretions (Taigen
Oftedal, 2002a); and the young of some live-bearing caecilians
feed on sloughed skin, skin secretions, or perhaps both
., 2006). The ‘crop milk’ of pigeons, ﬂamingoes
and emperor penguins is a lipid-rich material produced by
holocrine secretion by epithelial cells of the esophagus or crop
(Horseman and Buntin, 1995), but it does not match milk in
complexity, magnitude or duration of secretion. Lactation can
persist for many years in humans, great apes, toothed whales,
and elephants (West
., 2007), but may be as little as
3–4 days in the ice-breeding hooded seal (Oftedal
Lactation is highly complex and apparently of ancient evo-
lutionary origin (Oftedal, 2002b; Lefevre
., 2010). The
detailed signaling crosstalk among epithelial and underlying
mesenchyme cells that is required for the differentiation, ductal
branching, and proliferation of mammary tissue is only now
being unraveled (Watson and Khaled, 2008), and the func-
tional signiﬁcance and patterns of expression of thousands of
mammary genes are under investigation (Lemay
Secreted milk is extremely varied in composition – from trace
levels of fat in rhinos to more than 60% fat in some ice-breeding
seals (Oftedal and Iverson, 1995) – and contains unique
proteins (a-, b-, and k-caseins, b-lactoglobulin, a-lactalbumin,
whey acidic protein (WAP)), membrane-enclosed lipid droplets,
and sugars (lactose, milk oligosaccharides) that are not found
elsewhere in nature.
A dependency on milk is key to the life history strategy of
every mammal. Many mammals specialize on diets that
would be too difﬁcult for small offspring to capture or to
digest, or that might fail to cover the nutrient needs of
rapidly growing young if milk was not available (Pond,
1977). The ability to provide offspring with a highly diges-
tible, nutritionally balanced, and variably concentrated food
has enabled mammals to evolve a wide range of develop-
mental and reproductive strategies. Lactation provides the
essential nutrients required by the smallest and most altricial
of neonates, such as monotreme hatchlings and marsupial
neonates (Grifﬁths, 1978; Tyndale-Biscoe and Renfree,
1987), as well as the largest and most precocial of offspring,
such as hooded seals that only nurse for 4 days, but in
this time double their mass (Oftedal
., 1993a). Milk
production can be miniscule, as it must have been to feed
newborn Tasmanian tigers (or thylacines) which, by extra-
polation from living dasyuromorph marsupials, weighed less
than 40 mg at birth, equivalent to 0.00015% of the mass of
the 25 kg mother, surely the smallest birth mass : maternal
mass ratio of any mammal (O. Oftedal, unpublished results).
However, milk production can also be immense, as in the
100 000 kg blue whale. The pregnant female feeds in polar
and cold temperate waters where food is abundant and
where she lays down massive stores of blubber. She then
travels part way around the globe to warm or tropical areas,
gives birth to a calf, and while fasting or feeding a little,
produces an estimated 220 kg milk per day, containing
approximately 4000 MJ energy (Oftedal, 1997). Over the course
of the 6-month lactation, the blue whale cow transfers an
estimated 700 000 MJ to her calf, enough energy to feed
approximately 200 people for a year.
The ability to lactate allows mothers to collect nutrients
and deposit them in their tissues at one time and location,
but then provide these nutrients to their young as milk at
a later time and/or another location, such as in a nest or
burrow (rodents), cave (bats, bears) or snow den (polar bear)
or on an isolated island (sea lion, fur seal) or on ﬂoating rafts
of pack ice (various seals, walrus; Oftedal, 2000). Some
bears lactate in the winter den for 2 months before emerging
to feed and drink, a feat made possible by mobilization of
extensive body reserves, production of milk low in sugar
(minimizing the glucose demand of the mammary glands),
and recycling of water and perhaps nitrogen from cubs back
to the mother when she ingests their excreta (Oftedal
1993b). In bats, lactation must continue until pups have
deposited enough protein and mineral that their muscles
and bones have sufﬁcient strength to withstand the pressure
and torque associated with ﬂight, or the pups cannot feed
themselves; bat pups may individually reach 70% of maternal
mass before they are weaned (Hood
., 2011). In social
carnivores, an entire pack may hunt prey that is brought back
to the lactating female, who converts food constituents into
mammary secretions. The lactating female is in essence a ‘milk
factory’, rearing very large litters of rapidly growing young
(Oftedal and Gittleman, 1989).
How did such a remarkable ﬂuid as milk and such remark-
able biochemical factories as mammary glands evolve? The
mammary gland and its secretion represent major evolutionary
novelties, without any known intermediates. In the mid-19th
century the complexity and interdependence among mammary
glands, milk and dependent suckling young posed a challenge
to Charles Darwin’s theory of evolution by natural selection.
Darwin rose to the challenge, devoting most of a chapter of
the 1872 edition of
On the Origin of Species
to a discussion
of the problems of evolutionary novelty, such as the origin of
the eye and of the mammary gland. Believing that seahorse
eggs are hatched and reared in a brood pouch, Darwin (1872:
pp. 295–296) suggested by analogy that if mammalian young
were protected in a pouch containing cutaneous glands, a
gradual increase in the nutritive quality of the ﬂuid and of the
secretory complexity of the glands would be favored by natural
selection, leading ultimately to breasts producing milk. Over
the years since, a variety of authors have speculated on the
origin and evolution of lactation, trying to envision a process in
which something so complex could have evolved step by step
and be favored by natural selection (reviewed by Oftedal,
2002b). Unfortunately, most of these scenarios (including a
recent suggestion that lactation originated from the innate
immune system (Vorbach
., 2006; McClellan
have been put forth without reference to an evolutionary
timescale and do not acknowledge the repeated radiations
of increasingly mammal-like taxa since the ﬁrst appearance
of synapsids approximately 310 million years ago (mya).
Milk secretion in the context of synapsid evolution
The phylogenetic branch that would ultimately lead to
mammals (Synapsida) ﬁrst diverged from the branch leading
to ‘reptiles’ and birds (Sauropsida) in the mid-Pennsylvanian
period, approximately 310 mya. After this separation, there
were a series of sequential extinctions, with only a limited
number of taxa surviving into the succeeding geologic peri-
ods as the basis for future radiations (Sidor and Hopson,
1998; Oftedal, 2002b; Kemp, 2005). Thus, the basal synap-
sids radiated in the Pennsylvanian period and the early
Permian period; however, most lineages went extinct by the
mid–late Permian period, when they were succeeded by a
radiation of therapsids. Most therapsid taxa disappeared
during a massive extinction event at the end of the Permian
period, but cynodonts survived to radiate in the Triassic
period. Most of these lineages in turn disappeared in the late
Triassic period, but a subset – the mammaliaforms – radiated
in the late Triassic and Jurassic periods. It was from within
the mammaliaforms that true mammals evolved, perhaps in
the late Jurassic period, approximately 160 mya.
These sequential radiations incorporated an increasing
number of anatomic traits that now characterize mammals,
that is, they became progressively more mammal-like (Sidor
and Hopson, 1998; Kemp, 2005). Some of these traits involved
changes in locomotion (from a sprawled lizard-like gait to
an upright stance with improved running ability), in growth
pattern (from a periodic pattern of bone mineralization to more
continuous growth), in respiratory ability (development of
diaphragmatic breathing), in heat exchange (using respiratory
surfaces in the nasal cavity for cooling and moisture retention)
and in food processing ability (rearrangement of skull and
jaw bones for increased jaw musculature, diversiﬁcation
and specialization of teeth and tooth cusps). The picture is
one of increasing metabolic expenditure, increased growth
rates, increased activity, and a presumed upregulation of
basal metabolism and increased reﬁnement of temperature
regulation associated with endothermy.
Reproductive patterns also changed. The early synapsids
were egg-laying, like sauropsids (the lineage leading to
‘reptiles’ and birds) (Oftedal, 2002a). It was the development
of a complex of extraembryonic membranes to facilitate
respiration, use of stored yolk nutrients and segregation of
waste products within the egg that allowed the amniotes
(including sauropsids and synapsids) to venture onto land
and to gain a measure of independence from free water
(Packard and Seymour, 1997). However, unlike sauropsids,
the synapsids never evolved a calciﬁed eggshell, or at least
no fossil record of such has ever been discovered (in contrast
to the plethora of fossilized eggshells from dinosaurs,
crocodilians, turtles, birds and other sauropsids; Oftedal,
2002a). An egg with a parchment-like shell is the ancestral
condition of both groups; however, most sauropsids went
down a different evolutionary path, developing elaborate
eggshells of diverse crystalline form. These calciﬁed eggshells
are very resistant to vapor loss, having only small pores for gas
exchange. By contrast, the eggs with parchment-like shells
both gain and lose water rapidly. Eggs with parchment-like
shells are ectohydric, relying on environmental water for
completion of egg development, whereas eggs with calciﬁed
shells are by necessity endohydric, relying on water invested in
the egg before calciﬁcation and laying. Eggs with parchment-
like shells dry out rapidly when exposed to a vapor pressure
gradient, as in air that is not fully saturated with moisture, or
when exposed to a positive temperature gradient from egg to
environment (Oftedal, 2002a).
Early synapsids must either have buried their eggs in
moisture-laden soil, as do living lizards and snakes that
produce eggs with parchment-like shells, kept them hydrated
by contact with moist skin (as terrestrial-breeding frogs and
salamanders often do) and/or retained them within a moist
pouch, such as the egg-laying echidnas in Australasia. Yet
during the long period from the Pennsylvanian period to the
Jurassic period, the therapsids, cynodonts and mammaliaforms
are believed to have increased metabolic rates and elevated
body temperatures, and eggs were presumably incubated
to increase thermal stability (Oftedal, 2002a). A buried egg has
access to soil moisture, but is at soil temperature, whereas an
incubated egg in a nest is both isolated from soil moisture
and – due to thermal gradients that create vapor pressure
gradients – subject to rapid desiccation. Pythons that incubate
eggs with parchment-like shells above ambient temperatures
wrap tight body coils about the eggs to restrict moisture loss
., 2007). Some lizards and snakes overcome this
constraint by internal egg-retention, including development of
placental structures to provide nutrients to the developing
., 2006). However, early mammals
were still egg-laying (as indicated by egg incubation in extant
monotremes and the persistence of egg teeth in some
marsupial neonates; Oftedal, 2002a and 2002b).
It is not clear why the synapsids lineage did not evolve
egg-retention as a reproductive strategy, as this occurred
independently many times among sauropsids (Blackburn,
2006). Perhaps some radiations did, but went extinct without
leaving descendants. The synapsids that ultimately survived to
become mammaliaforms and mammals opted for a different
course: reduction in egg size and a switch to lactation as the
primary nutrient source for offspring.
Yet it seems impossible that the increasingly endothermic
synapsids could have incubated their eggs without facing
lethal egg dehydration unless there was an exogenous source
of moisture. I have argued that this required the evolution of
lactation, initially as a source of moisture for eggs (Oftedal,
2002a). Early synapsids apparently had glandular skin, and like
extant frogs and salamanders that rear terrestrial eggs, they
may have kept eggs moist via skin secretions (see ‘The evo-
lution of mammary secretion’ section). As eggs and hatchlings
were in contact with skin secretions rich in antimicrobial and
other protective compounds (see Whey Proteins section,
below), the evolution of nutrient-rich constituents in these
secretions could occur gradually and over a long time period.
Thus, milk was born.
It is not known whether eggs and hatchlings were kept in
a pouch, as Darwin had hypothesized, but this would have
Evolution of milk secretion
been an effective means of reducing moisture loss by eggs
and hatchlings. Many paleontologists have considered epi-
pubic bones as evidence that mammaliaforms and early
mammals (including early eutherians) carried eggs or young
in a pouch or suspended from the abdomen; however, others
argue that epipubic bones rather had a function in locomo-
tion as the structure of the pelvis evolved (Novacek
1997; Reilly and White, 2003). Pouches have evolved in
both monotremes (e.g. the echidnas) and marsupials (e.g.
kangaroos and wallabies), but these may not be of ancestral
origin; pouches appear to have been gained and lost multi-
ple times in marsupials (Tyndale-Biscoe and Renfree, 1987).
During the Triassic and the early Jurassic periods, the
cynodonts and mammaliaforms were not only developing
elevated metabolic rates but also were becoming progres-
sively smaller in size (Kemp, 2005). This miniaturization of
body size would have required miniaturization of eggs as
well. Even if eggs were kept in a pouch to prevent desicca-
tion, once they hatched the young would be too small to be
effective homeotherms (Hopson, 1973). In birds, reduction
of egg size in small species is accompanied by the reduction
of incubation time and hatching of altricial (incompletely
developed) young (Starck and Ricklefs, 1998). Hopson
(1973) argued that the diminutive mammaliaforms (some no
more than a few grams as adults; Luo
., 2001) must have
been producing altricial young, as small eggs could not hold
enough yolk to allow development of precocial (well devel-
oped) hatchlings. However, altricial young would require
feeding, indicating that lactation had already evolved.
This conclusion is bolstered by the fact that late cynodonts
and the mammaliaforms developed a reduction in tooth
replacement (Kemp, 2005). Early synapsids, like most saur-
opsids, had teeth that were replaced continuously as the
animal grew, allowing smaller teeth to be replaced by larger
teeth as the jaw lengthened with age. Most mammals, by
contrast, have only two sets of teeth, an initial set of
deciduous ‘milk teeth’ and the adult dentition. This devel-
opmental strategy, termed diphyodonty, is possible because
tooth eruption is delayed as the jaw develops
during the lactation period. There is no need for a robust,
adult-type teeth in dependent offspring that do not need to
capture or consume an adult-type diet. The fact that late
cynodonts and early mammaliaforms already had evolved
diphyodonty indicates that they were already reliant on milk
I conclude that the sequential radiations of basal synap-
sids, therapsids, cynodonts and mammaliaforms from the
Pennsylvanian period through the Jurassic period (i.e. from
approximately 310 to 160 mya) was a time of increasing
reliance on glandular skin secretions, to keep skin moist and
pliable, to provide water to eggs, and ﬁnally as a nutrient
source for offspring.
The evolution of mammary secretion
The mammary gland is a complex alveolar–ductal organ
involving a layer of secretory epithelial cells (lactocytes) that
secrete milk into the alveolus by a variety of mechanisms
(including exocytosis of secretory vesicles and budding out
and pinching off of milk fat globules, MFGs), a surrounding
layer of myoepithelial cells that expel milk from the alveolus
via contraction, and a basement membrane that surrounds
the epithelial compartment. The many alveoli are connected
to the skin surface by a much-branched ductal network that
terminates in canals, or galactophores, that penetrate either
thickened epithelial structures (the nipples) in eutherians
and marsupials or a specialized mammary patch or areola in
monotremes (Oftedal, 2002b).
The entire structure initiates development early in
embryonic life (before hatching from the egg in monotremes)
via coordinated reciprocal signaling between the epithelial
cells of ectodermal origin and the underlying mesenchyme of
mesodermal origin. This signaling directs gene expression,
morphogenesis of tissue architecture, and differentiation
of tissue-speciﬁc functions (Robinson, 2004; Watson and
Khaled, 2008). For example, signaling compounds generated
by the mesenchyme/stroma that have receptors in the
epithelial cells and that help determine ductal and alveolar
development include hepatocyte growth factor (HGF), IGF-1,
activin/inhibin B, epimorphin, neuroregulin and keratinocyte
growth factor (also called ﬁbroblast growth factor-7; Nelson
and Bissell, 2006). This type of branching morphogenesis
driven by epithelial–mesenchymal interactions and involving
coordinated development with stimulatory signaling in
part from HGF and epidermal growth factor, balanced by
inhibitory signaling from members of the transforming
growth factor-bfamily, is also found in tissues of even more
ancient evolutionary origin, such as the pancreas, lung,
kidney, prostate and salivary glands (Nelson and Bissell,
2006). Thus, the developmental pathways of the mammary
gland must derive from some pre-existing tissue, presumably
glandular tissue associated with the skin, which was
co-opted for a new function, the secretion of a nutritive ﬂuid
for feeding of the young. Although the basic pattern of
mammary development, and its regulation, may derive from
a more ancient model, the extent of glandular proliferation
and output – the remarkable repeated cycles of proliferation
and secretion followed by cellular apoptosis and gland
involution – and the types of secretory products formed
represent evolutionary novelties.
The synapsid lineage that led to mammals derived
from pre-amniotic tetrapods, a group now represented by
living amphibians (termed lissamphibia by paleontologists).
Amphibian skin is characterized by a relatively thin epidermis
containing a few cell layers of stratum corneum and dense
coverage of small multicellular secretory glands that have
evolved to secrete primarily mucus (mucous glands) or
bioactive constituents (granular glands) onto the skin sur-
face (Clarke, 1997). Both gland types apparently secrete via
exocytosis of secretory vesicles; however, secretory cells in
granular glands also swell with contents and release bulk
material into the lumina of the glands via a holocrine process.
This material may then be discharged onto the skin surface
by contraction of myoepithelial cells when the animal is
agitated or stressed. Synapsids apparently inherited a gland-
ular skin from the tetrapods. A remarkable early Permian
fossil of the integument of the therapsid
(Dinocephalidae) includes a dense pattern of concave lens-
like structures; Chudinov (1968) interpreted these as multi-
cellular, ﬂask-shaped alveolar glands – similar to the glands
of amphibian skin – and argued that a glandular skin is a
primitive synapsid feature still evident in mammals. It should
also be recognized that the complex combinations of a- and
b-keratins, multi-layered scales and paucity of skin glands in
living reptiles evolved after the separation of sauropsids and
synapsids and is not ancestral to the mammalian integument
However, could such glands evolve into mammary glands?
On the basis of evidence that elements of the innate immune
system are incorporated into milk constituents and that
certain signaling pathways of the innate immune system also
have a role in regulating mammary development, Vorbach
. (2006) speculated that mammary secretion ﬁrst
developed as part of an inﬂammatory response by mucous
secreting cells, and McClellan
. (2008) supported this
view. The innate immune system certainly has an ancient
origin, including the antimicrobial compounds associated
with epithelial structures and secretions (Beutler, 2004).
In frogs and other amphibians, mucous glands are important
to keep the skin surface moist, facilitating exchange of
respiratory gases across the very thin epidermis (Lillywhite,
2006), and amphibian cutaneous glands are indeed veritable
factories of antimicrobial constituents. At least 500 anti-
microbial peptides have been found in amphibian skin
glands to date (Jenssen
., 2006). However, mucous and
granular glands are very different in structure and secretion
from mammary glands. In mammals, mucous glands are
restricted to oral and internal epithelial surfaces. Although
. (2006) are certainly correct that mammary
glands must derive, ultimately, from the simple glandular
skin structures found in pre-amniotic tetrapods (Quagliata
., 2006), there must have been intermediate stages that
more closely resemble mammary glands.
If one compares mammary glands to other mammalian skin
glands, they bear close resemblance to only one type of gland,
the apocrine glands on the general skin surface of mammals
(Oftedal, 2002b). Apocrine glands and mammary glands
secrete constituents both by exocytosis of secretory vesicles
and by a budding out and pinching off of cellular contents
with loss of cytoplasm. In apocrine glands, the latter process
is considered an apocrine mode of secretion, in contrast to
merocrine secretion employing exocytosis of vesicles or holo-
crine secretion in which cells swell with the secretory product
that is released via apoptotic disruption of cellular integrity. In
mammary glands, budding out and pinching off occur during
secretion of MFG; cytoplasmic crescents may be present but
are minimal (Mather and Keenan, 1998; Mather, 2011). It is
likely that MFG secretion is a highly derived form of apocrine
secretion in which upregulation of milk fat secretion has
required incorporation of novel membrane constituents (see
Milk Fat Globule section, below). Unfortunately, little is known
about the details of secretion in generalized apocrine glands or
about the genes expressed and proteins synthesized (Oftedal,
2002b), although at least some types do not produce milk-
speciﬁc proteins, such as b-casein (Gritli-Linde
From an evolutionary perspective, it would be very interesting
to compare the array of genes expressed by developing
and secreting apocrine glands with those expressed during
mammary gland development, milk secretion, and mammary
involution. For example, nearly 200 milk protein genes and
more than 6000 other genes have been identiﬁed as expressed
in the mammary glands in virgin, pregnant, lactating, involut-
ing, and mastitic cows (Lemay
., 2009), but how many
of these genes are expressed in apocrine glands is unknown.
It would also be instructive to compare gene expression of
both apocrine and mammary glands with that occurring in
amphibian skin glands to document similarities and differences
that may indicate ancestral and derived conditions.
In most mammals, an apocrine gland on the general skin
surface is typically associated with both a hair follicle and a
sebaceous gland in a triad termed an apo-pilo-sebaceous
unit (APSU). The development of the APSU occurs in coor-
dinated fashion, no doubt because of crosstalk between the
differentiating epithelial cells and underlying mesenchyme,
as well as differences in signaling pathways and receptors
of the hair follicle, apocrine gland, and sebaceous gland
(Hatsell and Cowin, 2006; Andrechek
., 2008; Mayer
., 2008). The apocrine gland duct typically opens into
the infundibulum of the hair follicle, such that secretion
contacts the hair shaft. Remarkably, in monotremes, a similar
relationship exists between mammary glands, hair follicles,
and sebaceous glands; the three form what can be termed
a mammo-pilo-sebaceous unit (MPSU; Oftedal, 2002b). The
galactophores (lactiferous ducts) also open up into the infun-
dibula of enlarged, specialized mammary hairs (Grifﬁths,
1978). The mammary glands in monotremes are organized into
a small oval mammary patch or areola consisting of 100 to 200
MPSUs (Grifﬁths, 1978; Oftedal, 2002b); there is no nipple.
In the area surrounding the mammary patch, APSUs develop.
The mature, lobular mammary gland in mid-to-late lactation
is very much larger, more branched, and contains many
more secretory epithelial cells than an apocrine gland, but in
earliest lactation — when monotreme eggs are incubated and
hatched – the mammary gland is still relatively small and
tubular (Grifﬁths, 1978), and thus has a superﬁcial resemblance
to an apocrine gland.
In marsupials, such as opossums and kangaroos, there
is also a developmental association of mammary glands
with hair follicles and sebaceous glands. According to an
early work by Bresslau (1912 and 1920), an oval primary-
primordium separates into nipple primordia, which deepen
into knobs and bud out into hair follicles (primary sprout),
mammary glands (secondary sprout) and sebaceous glands
(tertiary sprouts). In the opossum, for example, eight hair
follicle sprouts are associated with eight mammary sprouts
and eight sebaceous sprouts; that is, the nipple primordium
develops into eight MPSUs. The hair follicles penetrate the
nipple epithelium during development, but are subsequently
Evolution of milk secretion
shed, each leaving a duct (galactophore) by which the mam-
mary gland communicates to the surface of the nipple. As
opossums have a dozen or more nipples, approximately 100
MPSUs are involved. In the adult marsupial, the ‘mammary
hairs’ are no longer evident, but the galactophores bear testi-
mony to their previous existence. Among different species, the
number of ducts penetrating the nipple reﬂects the numbers of
primary hair follicle buds, and can vary from 3 to 33 (Tyndale-
Biscoe and Renfree, 1987); however, in species with large
numbers of presumptive MPSUs, it is not certain that all
secondary sprouts develop into functional mammary lobules.
In eutherian mammals, the association of apocrine glands
with hair follicles (APSUs) is maintained, but the association
of mammary glands with hair follicles – the presumed ancestral
condition – appears to have been lost. In 2002, I hypothesized
that this must be due to the inhibition of hair follicle develop-
ment in the vicinity of mammary glands, and suggested that if
the presumptive inhibiting compound (s) could be blocked at
the earliest stages of mammary development, hair follicles may
develop in association with mammary buds (Oftedal, 2002b).
Although the actual signaling pathways are undoubtedly com-
plex, with both shared and differing sensitivities to signaling
compounds among different epithelial cell types, it is intriguing
that bone morphogenetic proteins (BMPs) inhibit hair follicle
formation, and that when Mayer
. (2008) reduced BMP
signaling in the mouse by transgenic overexpression of a
BMP antagonist, nipple epithelium was converted into pilo-
sebaceous units. Mayer
. (2008) hypothesized that the BMP
pathway had been co-opted during evolution of the nipple to
suppress hair follicle formation. If so, this occurred following the
separation of marsupials and eutherians, given that hair follicle
development is not suppressed during nipple development in
marsupials; rather, hair follicles subsequently regress and the
hairs are shed. Bresslau (1920) even described a developmental
stage in the koala, after the nipple has everted (extended above
the skin surface), in which a tuft of mammary hairs protrude
through galactophores at the apex of the nipple.
Thus, available evidence is consistent with the hypothesis
that mammary glands developed from an apocrine-like gland
that had an association with hair follicles and sebaceous
glands. This is not in conﬂict with the Vorbach
hypothesis that mammary gland secretion had its origin in
mucous glands, as the apocrine-like glands themselves must
have originated from earlier tetrapod cutaneous glands. The
innate immune system itself is of even more ancient origin,
with components shared among invertebrates and vertebrates
(Beck and Habicht, 1996; Hoffmann
., 1999; Fujita, 2002),
and thus the co-option of innate immune system components
into the regulatory elements of epithelial–mesenchyme sig-
., 2008) probably occurred long before
the origin of mammary glands.
The evolution of milk constituents
The secretory products of the mammary gland represent the
expression of a large number of genes that are upregulated
during lactation, but many of these products remain
unknown (except as genes) or their functionalities are poorly
., 2007; Lemay
., 2009). Com-
parisons of monotreme, marsupial and eutherian genomes
suggest that milk and mammary genes tend to be conserved,
that is, have not diverged as much as other genes (Lemay
., 2009). Nonetheless, there are major constituents of milk
in these three lineages for which comparative data provide
evidence of an evolutionary origin, especially when milk pro-
teins are compared with closely related gene products.
All mammalian milks that have been studied contain multiple
casein proteins, characterized as a-, b-andk-caseins. The
caseins are phosphorylated during synthesis, and aggregate
into large micelles containing calcium bound to phosphorus in
calcium phosphate nanoclusters (Smyth
., 2004). Multiple
caseins participate in these micelles; however, k-casein plays
a particularly important role in stabilizing the micelle in
secreted milk. The caseins provide a large proportion of the
amino acids, calcium, and phosphorus transferred to the
young, and via curd formation in the neonatal stomach they
play a part in fat and protein digestion. The caseins had
already diverged into the three primary types, a-, b-, and
k-caseins, before the separation of monotremes, marsupials
and eutherians, as each casein type is found in all three taxa
(Rijnkels, 2002 and 2003; Lefevre
., 2009 and 2010). Thus,
caseins have a pre-mammalian origin.
The caseins are members of a much larger family of pro-
teins of unfolded nature that are secreted from cells, usually
in association with tissue mineralization or regulation of
calcium at target tissues. These proteins, termed secretory
calcium-binding phosphoproteins (SCPPs), are secreted by
secretory epithelial cells or cells derived from underlying
ectomesenchymal cells, and have an ancient history in the
evolution of mineralized vertebrate tissues (Kawasaki and
Weiss, 2003; Kawasaki, 2009). As unfolded proteins, all
SCPPs are low in cysteine and therefore cystine disulﬁde
bridges, and a subclass of the proteins (P/Q-rich SCPPs),
including the caseins, are particularly rich in proline and
glutamine (Kawasaki and Weiss, 2003; Kawasaki
2011). On the basis of the relative locations and structures of
exons of these P/Q-rich SCPPs, as well as their phylogenetic
. (2011) proposed that the a- and
b-caseins derived via gene duplication and exon changes
from an ancestral gene (
) that derives from another
(which itself is derived
), whereas k-casein derives from the
(which is also derived from
Many P/Q-rich SCPPs, including the
derived proteins, are expressed in mammalian ameloblasts
and are involved in mineralization of tooth enamel; follicular
dendritic cell secreted peptide (from
) is found in soft
connective tissue (periodontal ligament), where it is thought
to prevent spontaneous precipitation of calcium phosphate;
it is also expressed in the mammary gland (Kawasaki, 2009;
., 2011). Kawasaki
. (2011) suggest that
the initial function of an ancestral SCPP (probably a k-casein
precursor) in protolacteal secretion may have been to reg-
ulate calcium delivery to the surface of an egg and to prevent
precipitation of calcium phosphate on the parchment-like
. (2011) hypothesize that this may
have occurred before the divergence of sauropsids and
synapsids, although an ancestral
has yet to be found
in a sauropsid genome.
Subsequently, the types of caseins, and also the numbers
of genes involved in producing each type, increased via gene
duplication and exon changes (Rijnkels, 2002; Lefevre
2009 and 2010). By an unknown evolutionary process, the
different caseins became involved in the formation of com-
plex micelles stabilized by calcium and phosphate bonds
., 2004). This transformation of ancestral SCPPs
into a complex of micelle-forming proteins was essential in
converting milk from an egg supplement to a major source
of nutrients for the suckling young. Given the small size of
mammaliaforms in the late Triassic and Jurassic periods, and
hence the small size of their eggs (Hopson, 1973; Oftedal,
2002b), the novel nutritive function of these SCPPs must
have developed before this time, for example, during the
Permian and Triassic periods. This is consistent with the
estimated loss of multiple vitellogenin genes beginning
approximately 170 mya in the Jurassic period (Brawand
., 2008). Vitellogenin genes could only be inactivated
once the nutrient transport function of the caseins had made
egg yolk proteins dispensible.
The Milk fat globule (MFG)
Mammals vary tremendously in the fat content of their milk
(from less than 1% in rhinos and some lemurs to 60% in
some seals; Oftedal and Iverson, 1995); however, in all
species studied, milk lipids are secreted as specialized
structures known as MFGs. MFGs are lipid spheres bounded
sequentially by a phospholipid monolayer, an inner protein
coat, a bilayered phospholipid membrane, and a glycosy-
lated surface (Mather and Keenan, 1998). The collective term
for the multi-layered structure or envelope that encloses the
lipid sphere is the milk fat globule membrane (MFGM),
which is a structure found only in milk. The method of milk
lipid secretion appears to be unique, as it has not been found
in other organs (McManaman
., 2006); if this general-
ization withstands further investigation of lipid secretion
in other gland types, the MGM must be considered a key
evolutionary novelty of lactation.
What is of particular interest is that the MFGM contains
proteins that appear essential to the synthesis and secretion
of MFGs. In particular, two proteins, butyrophilin and xan-
thine oxidoreductase (XOR), play an obligatory structural
role in MFGM synthesis, and if they are reduced or elimi-
nated from mouse mammary cells via knockout of the genes
that code them, mice fail to produce normal milk; the tri-
acylglycerols within the secretory cells fail to be secreted into
milk fat droplets, but rather accumulate in the cytoplasm or
leak into the alveolar lumen as unstructured, amorphic lipid
., 2002; Ogg
., 2004). Although the
details of protein–protein interactions during formation of the
MFGM are not fully understood (Mather, 2011), these two
proteins have apparently been co-opted from other cellular
functions during the evolution of the mammary gland.
The butyrophilin in milk is now correctly speciﬁed as
butyrophilin1A1, as it is the gene product of only one of the
) that code for the family of proteins known as
., 2001). The butyrophilins are
part of the immunoglobulin superfamily and contain two
folded immunoglobulin domains, a transmembrane domain
and a C-terminal end that may include a large B30.2 domain.
In addition to its role in the MFGM, butyrophilin1A1 has been
found to be expressed within the thymus; other butyrophilins
are more widely expressed among tissues (Smith
2010a). Butyrophilins and related proteins of the immunoglo-
bulin superfamily appear to play a role in the regulation of
proliferation, cytokine secretion and activity of T-cells, and
butyrophilin1A1 retains this function, at least
., 2010a). It is interesting that butyrophilin1A1 is the only
butyrophilin that appears to bind XOR via its B30.2 domain,
and it is this binding that is believed to be critical to MFG
secretion from lactocytes (Jeong
., 2009). It appears that
the ancestral butyrophilin protein was a transmembrane pro-
tein in secretory cells that had functions in local immune
response, and subsequently evolved a role in synthesis and/or
stabilization of the MFGM.
Xanthine oxidoreductase, or XOR, is best known for its role
in catalysis of the last two steps in the formation of uric acid,
an important nitrogenous waste product, but it has multiple
enzymatic functions, and is a member of the molybdo-
ﬂavoenzyme (MFE) protein family (Garattini
., 2003). The
MFEs are believed to have evolved as an ancestral XOR in
., 2003). Although the XOR gene is
sometimes considered to code for a housekeeping protein
., 2002), this is debatable, as XOR is unequally
expressed in cells (Garattini
., 2003). In mammals, XOR is
initially synthesized as the enzyme xanthine dehydrogenase,
but it is readily converted to xanthine oxidase, which is the
form typically recovered from milk (Enroth
., 2000; Nishino
., 2008). Xanthine oxidase generates free radical and
reactive nitrogen species, and is upregulated and appears to be
during inﬂammation, leading to the hypotheses that XOR has
important antimicrobial activities, perhaps even in milk (Martin
., 2004), and that XOR may have had an important role in
the evolution of innate immunity (Vorbach, 2003). Certainly,
XOR and innate immunity are both of pre-eukaryote origin,
and were important long before mammary glands evolved. Yet
the upregulation and apical membrane localization of XOR in
mammary epithelial cells during mammary gland development
., 2002); the binding of XOR to the B30.2
domain of butyrophilin (Jeong
., 2009); and the failure of
MFG formation in heterozygous XOR knockout mice (Vorbach
., 2002) all indicate a novel function for XOR in the
MFGM. Other MFGM components, such as adipophilin, also
have important, if incompletely understood, functions (Mather,
2011), and each of these has an evolutionary history.
MFG secretion presumably evolved from some previous form
of fat secretion, perhaps by tetrapod or synapsid skin glands.
Evolution of milk secretion
Certainly, some extant frogs secrete lipids as a means of
reducing water loss across the skin (Lillywhite
Lillywhite, 2006), and secreted lipids applied to eggs could
have had an impact on egg moisture loss (Oftedal, 2002a).
However, much more research is required to understand the
differences and similarities of secretory mechanisms. Mam-
mary glands bear developmental and structural resemblance to
apocrine glands, which led to the hypothesis that mammary
glands are derived from ancient apocrine-like glands (Oftedal,
2002b). One can imagine a scenario in which an ancestral
apocrine secretion entailed the secretion of apical blebs con-
taining cytoplasm, secretory vesicles and perhaps cytoplasmic
lipid droplets, similar to the process described for some
specialized apocrine glands, such as human axillary apocrine
glands, glands of Moll, ceruminous glands in the outer ear
canal, and rodent Harderian glands (Gesase and Satoh, 2003;
., 2003; Stoeckelhuber
., 2011). With an upregulation of mammary
fat synthesis, mechanisms may have evolved to minimize
cytoplasmic loss to a few cytoplasmic crescents, as in milk
secretion. The increase in lipid secretion must have occurred
before the miniaturization of the mammaliaforms in the
Triassic period and gradually replaced the nutritional role of
lipids provided by yolk, allowing the inactivation of vitello-
genins involved in transport and storage of lipids in the egg
Milk sugar synthesis
All mammalian milks contain at least traces of sugar (Oftedal
and Iverson, 1995); in most eutherians the predominant
sugar is lactose, whereas in monotremes, marsupials and some
eutherian carnivores oligosaccharides predominate (Urashima
., 2001; Messer and Urashima, 2002; Uemura
., 2010). Both lactose and oligosaccharides with
lactose at the reducing end are unique to milk (Toba
1991) and require a novel synthetic pathway.
In the mammary secretory cell, the synthesis of lactose begins
with the synthesis of a unique milk protein, a-lactalbumin,
in the rough endoplasmic reticulum (Brew, 2003). a-lactalbumin
is then transported to the Golgi apparatus. A transmembrane
protein in the trans Golgi, b-1,4-galactosyltransferase1
(b4gal-T1), binds UDP-galactose, producing a conformational
change that allows a-lactalbumin to be bound (Ramakrishnan
and Qasba, 2001). When a-lactalbumin binds to b4gal-T1,
it alters the speciﬁcity of b4gal-T1, allowing glucose to
become the acceptor sugar for galactose transfer, resulting
in the synthesis of lactose. Thus, a-lactalbumin acts as a
regulator of b4gal-T1, and without a-lactalbumin b4gal-T1
does not synthesize lactose under physiological conditions
It has been apparent for many years – from amino acid
sequence similarity, three-dimensional structure and the
structure of the exons that code for a-lactalbumin – that
a-lactalbumin is most closely related to c-type lysozyme and
is derived from it via gene duplication and base pair substi-
tution (Prager and Wilson, 1988; Qasba and Kumar, 1997;
Brew, 2003). The estimated date of origin of a-lactalbumin
from c-lysozyme is ancient, before the time of the split of
synapsids from sauropsids approximately 310 mya (Prager
and Wilson, 1988). Many authors have been puzzled by this
date, as it was assumed that mammary glands did not arise
until the appearance of ‘early mammals’ (i.e. mammaliaforms)
100 million or more years later (Hayssen and Blackburn, 1985;
Prager and Wilson, 1988; Qasba and Kumar, 1997; Messer
and Urashima, 2002), but it is consistent with an ancient
origin of lactation.
It is probable that c-lysozyme, as a normal antimicrobial
constituent of epithelial secretions and egg white (Callewaert
and Michiels, 2010), would have been present in tetrapod
and early synapsid skin secretions, including secretions
delivered to eggs; a c-type lysozyme is present in amphibian
skin secretions (Zhao
., 2006). Although the anti-
microbial function of c-lysozyme would presumably help
protect eggs (as it does in egg white), what immediate
advantage would accrue to eggs or hatchlings from the
conversion of c-lysozyme function to that of a-lactalbumin,
with the resultant synthesis of lactose? a-lactalbumin does
not have lysozyme activity, whereas lysozymes do not
bind to b4Gal-T1, because of differences in amino acid
composition at key positions involved in binding of substrate
(in lysozyme) or binding of b4gal-T1 (in a-lactalbumin;
Ramakrishnan and Qasba, 2001; Messer and Urashima,
2002; Brew, 2003; Callewaert and Michiels, 2010), and thus
an ‘intermediate’ with both functions may not have been
possible. In addition, one must assume that any lactose thus
synthesized would have been indigestible to embryos or
hatchlings, given that the intestinal brush-border enzyme
lactase could not have evolved without a substrate to digest,
and lactose does not occur elsewhere.
Messer and Urashima (2002) argue that the ancestral
function of a-lactalbumin as a b4Gal-T1 regulator may
have been the production of lactose-containing free oligo-
saccharides, rather than free lactose
. The amount of
a-lactalbumin synthesized in the monotreme mammary
gland is minor, and Messer and Urashima (2002) assume this
to be the ancestral condition. A wide range of glycosyl-
transferases would have been present in the trans Golgi of
tetrapods and early synapsids, as these are part of the nor-
mal synthetic machinery for glycosylation of glycoproteins,
glycolipids, and proteoglycans in vertebrates, and they are of
ancient origin (Varki, 1998; Lowe and Varki, 1999). A low
rate of lactose synthesis, coupled with high activity of other
glycosyltransferases that could glycosylate lactose, may have
produced free oligosaccharides rather than free lactose,
similar to what is observed in extant monotremes and mar-
supials. Milk oligosaccharides have antimicrobial or probiotic
effects, for example by leading pathogens to ‘mistake’ free
oligosaccharides for the oligosaccharide chains of the gly-
cocaylx on apical cell membranes (Newburg, 1996), and thus
to fail to bind to these surfaces. Such an effect might beneﬁt
the mammary gland, an egg surface, or the digestive tract of
a hatchling even before the evolution of the lactase enzy-
matic mechanism. It is intriguing that marsupial young that
consume milk containing oligosaccharides but not lactose do
not have intestinal lactase (Crisp
., 1989), but whether
this is the ancestral mammalian condition is not known.
In eutherians, lactose accumulating in the Golgi apparatus
creates an osmotic gradient, which draws water into the
Golgi; this aqueous phase (including lactose, a-lactalbumin,
other whey proteins, caseins, electrolytes, etc.) is subsequently
packaged into secretory vesicles for transport to the apical
plasma membrane of the secretory cell (Shennan and Peaker,
2000). This model of milk secretion entails substantial
upregulation of a-lactalbumin and lactose synthesis, and the
transcription of b4Gal-T1 is also upregulated above con-
stitutively expressed levels via the use of a second transcrip-
tional start site, regulated by a stronger promoter and by more
efﬁcient translation of the truncated transcript (Shaper
1998). Although long considered the ‘standard’ model of milk
secretion, this may represent a derived feature of eutherian
lactation that only developed after the young evolved the
ability to digest lactose. One group of eutherian mammals, the
fur seals and sea lions (Pinnipedia: Otariidae), have secon-
darily lost the ability to synthesize a-lactalbumin because of
gene mutations and changes in transcription rates (Sharp
., 2005; Reich and Arnould, 2007; Sharp
therefore the milk is devoid of lactose or lactose-based
., 1987a and 2011). These taxa
manage to produce large volumes of high-fat milk (Oftedal
., 1987b; Arnould and Boyd, 1995; Arnould
but the secretory processes by which the aqueous phase is
secreted have not been studied. In mice, knockout of the gene
for a-lactalbumin results in a very low level of secretion
of high-fat milk, and the offspring do not survive (Stinnakre
., 1994; Stacey
Whey proteins as amino acid sources
Caseins have a loosely folded structure with few cystine dis-
ulﬁde bonds, and as a consequence contain a relative deﬁcit
of sulfur-containing amino acids (SAA, i.e. methionine and
cysteine) relative to the requirements of offspring. In cow’s milk,
-, b-, and k-caseins contain approximately 2.9% to 3.7%
SAA, by mass, whereas a-lactalbumin and b-lactoglobulin
presented in Fox 2003). Suckling mammals appear to require
that SAA be present as 4% to 6% of total amino acids in order
to attain maximal growth (Foldager
., 1977; Burns and
Milner, 1981; Fuller
., 1989; National Research Council,
1995). Methionine can substitute for cysteine in most cases
(except, perhaps, in premature human infants; Fomon
., 2008), but cysteine can only replace
approximately half of the methionine requirement in growing
., 1989). In formulating casein-based diets,
supplemental cysteine or methionine are required to compen-
National Research Council, 1995). This suggests that other
balanced source of amino acids, rather than just a supplement.
The major milk-speciﬁc whey proteins, depending on species,
are a-lactalbumin (e.g. in human milk), b-lactoglobulin (e.g. in
cow’s milk), and WAP (e.g. in rat milk), but additional whey
proteins have been identiﬁed in marsupials, such as early lac-
tation protein, late lactation protein, and trichosurin (Nicholas
., 1987; Piotte and Grigor, 1996; Demmer
b-lactoglobulin is the major whey protein in most ruminant
milks (including dairy animals such as dairy cattle, goats,
sheep and water buffalo), but does not have any indisputable
biological role beyond supplying amino acids to the offspring
(Sawyer, 2003). As b-lactoglobulin occurs in the milks of
monotremes (platypus), several marsupials (brushtail possum,
wallabies and kangaroos) and at least 35 species of euther-
ians, it must have evolved before the divergence of these
groups in the Jurassic or Cretaceous period. The discovery that
b-lactoglobulin was similar in structure to retinol-binding
protein (RBP) led to the hypothesis that b-lactoglobulin might
have a role in the transport to the young of vitamin A, vitamin
D, fatty acids, or some other essential lipophilic compounds, or
may play a role in intestinal uptake of these constituents
(Pervaiz and Brew, 1985; Perez and Calvo, 1995; Yang
2009). However, in ruminants, vitamin A is associated with
the fat globule and not b-lactoglobulin; in pigs and horses,
b-lactoglobulin does not bind either retinol or fatty acids;
and, in suckling pups of mice that have not been genetically
modiﬁed as per Yang
. (2009), vitamin D is obviously
absorbed from milk despite the absence of b-lactoglobulin
or the pups would develop vitamin D deﬁciencies. Thus, if
b-lactoglobulin has any role in transport and/or intestinal
uptake of these lipophilic constituents, it is neither essential
nor universal (Perez and Calvo, 1995). A major problem in
ascribing a functional role is that b-lactoglobulin is absent
from the milks of so many mammals, including laboratory mice
and rats, guinea pigs, domestic rabbits, dromedary camels,
llamas and humans (Sawyer, 2003).
Both b-lactoglobulin and RBP are members of a large
family of small extracellular proteins, termed lipocalins, that
have similar tertiary structure, speciﬁc amino acid sequence
motifs and exon–intron structure of coding genes (Flower,
., 2006). This ancient protein family (or
superfamily) apparently derives from a bacterial protein and is
characterized by a barrel-shaped lipophilic cavern surrounded
by a series of eight b-strands, and that is open on one
., 2006). Many lipocalins are known to
function via transport and/or sequestration of hydrophobic
compounds in this ‘barrel’ and occasionally at secondary bind-
ing sites. Analysis of the molecular evolution of the lipocalins
suggests that RBP diverged from the other lipocalins ﬁrst,
followed by b-lactoglobulin, suggesting that b-lactoglobulin is
of more ancient origin than other vertebrate lipocalins (other
than RBP), including lipocalins that are found in ﬁsh and
., 2000; Sanchez
., 2003 and
2006). It is likely that the ancestral b-lactoglobulin had similar
function to that of an ancestral RBP-like protein, that is, trans-
porting hydrophobic compounds in extracellular and/or secre-
ted ﬂuids, long before the appearance of milk as we know it.
Although b-lactoglobulin retains a generalized ability to bind a
variety of hydrophobic ligands, because of the elasticity of the
outer parts of the barrel (Konuma
., 2007), its current role in
Evolution of milk secretion
milk appears to be primarily a nutritional one, and in species in
which other whey proteins predominate, b-lactoglobulin has
become superﬂuous and has been lost. Two b-lactoglobulin
genes have been observed in ruminants, but one is non-coding
and is thus a pseudogene (Sawyer, 2003). A b-lactoglobulin
pseudogene is also suspected in the human genome, but
there may be confusion with the glycodelin gene (Kontopidis
., 2004). Other lipocalins (trichosurin, late lactation
protein) are expressed in marsupial milk (Demmer
., 1998); however, these are only distantly related
to b-lactoglobulin and are apparently of more recent origin
Among whey proteins, WAP has the highest sulfur amino
acid content, approximately 17% to 20% by mass. The key
feature of WAP is the presence of two or three domains of
approximately 40 to 50 amino acids, each of which contains
eight cysteine residues involved in four disulﬁde bonds; as
the domain was ﬁrst recognized in WAP, it is termed the
Whey Acidic Protein Four-Disulphide Core (WFDC) domain.
There are at least 33 distinct (non-homologous) proteins
among vertebrates and invertebrates that include one to
four WFDC domains (see PROSITE, www.expasy.org/cgi-bin/
prosite), including proteins with antibacterial, antiviral and
anti-inﬂammatory functions, as well as several proteinase
inhibitors. All are secreted proteins, including proteins in
respiratory, reproductive and other epithelial secretions
., 2003; Bingle
., 2006). The structural
similarity of WAP to other WFDC-containing proteins has led
to speculation that WAP may also have antibacterial or
proteinase inhibition functions, but attempts to demonstrate
this have failed (Hajjoubi
., 2006; Sharp
There is evidence that WFDC domains inﬂuence cell pro-
liferation and growth
and in transgenic mice
(reviewed by Topcic
., 2009), but when the WAP gene is
deleted in knockout mice, the mice continue to develop
normal mammary glands indicating that WAP is not essential
for mammary cell differentiation or proliferation (Triplett
., 2005). The primary effect of WAP deletion in mice
appears to be growth retardation of the young during the
second half of lactation. Thus, the functional role of WAP in
milk, other than as a rich source of sulfur amino acids for the
young, remains unclear.
The WFDC domain itself is of ancient origin, being a
component in secreted proteins involved in the regulation of
shell mineralization in mollusks such as abalone (Treccani
., 2006), and in antimicrobial response as part of the
innate immunity of crustaceans and perhaps insects (Zou
., 2007; Jia
., 2008; Smith
., 2010b). A number
of WFDC domain-containing proteins are also secreted by
snake venom glands, where they have antibacterial function
., 2007; Fry
., 2008), and by skin glands in
frogs, where they serve as antimicrobial defensive com-
., 2002; Zhang
., 2009). Although much
more research is required to determine relationships among
invertebrate and vertebrate WFDC-containing proteins, it is
likely that an ancestral WAP present in the glandular skin
secretion of an egg-tending tetrapod or early synapsid
served as a defensive compound against microbes as a
component of the innate immune system, similar to existing
WFDC proteins in mammalian epididymal, respiratory, and
oral mucosal secretions (Hiemstra, 2002; Hagiwara
2003; Bingle and Vyakarnam, 2008), and in frog skin secre-
., 2002; Zhang
As with b-lactoglobulin, it appears that WAP may have
lost its purported ancestral function as proteinase inhibitor/
antimicrobial protein in skin secretions. Of the two WFDC
domains in WAP in eutherian milks, designated as DI
and DII, only DII retains the characteristic N-terminal motif
found in most WFDC domains (Lys-X-Gly-X-Cys-Pro, where X
represents various amino acids); amino acid substitutions in
this and other areas of DI may have altered the charge dis-
tribution, glycosylation sites, and conformation in such a way
that original functions are no longer possible (Ranganathan
., 1999). Monotreme and marsupial WAPs contain two
to three WFDC domains, but of differing sequence and
arrangement than eutherian WAPs (Sharp
., 2007), and
it is thought that they may retain functions lost in eutherians,
but more evidence is required (Topcic
., 2009). At least
some eutherians have lost WAP in entirety. Although the
genes for WAP synthesis have been found in sheep, goats,
and cattle, they are missing a nucleotide at the end of the
ﬁrst exon, causing a frameshift mutation (Hajjoubi
2006). They are not transcribed and are thus pseudogenes.
The proposal that mammary secretion has an ancient origin
and long evolutionary history (Oftedal, 2002a and 2002b) is
increasingly accepted, especially in the face of supportive
molecular evidence (Kawasaki and Weiss, 2003; Vorbach
., 2006; Brawand
., 2008; McClellan
Capuco and Akers, 2009; Lemay
., 2009; Lefevre
., 2011). It is now possible to formulate
a more detailed scenario by which the secreted ﬂuid came to
resemble in form and function what we now know as milk.
The evolutionary origins of milk appear to be found in the
secretions of primitive apocrine-like glands in the skin of
early synapsids or even of taxa living before the split of
synapsids from sauropsids approximately 310 mya. These
glands incorporated elements of the innate immune system
in providing protection to the skin and to eggs that were
moistened. Membrane and intracellular proteins in secretory
epithelial cells, such as butyrophilin and xanthine oxidase,
were incorporated into the MFGM, secretory calcium-bind-
ing phosphoproteins involved in extracellular regulation of
calcium and phosphorus were transformed into casein
micelles, and antimicrobial proteins such as lysozyme and
WFDC-containing proteins were incorporated as whey pro-
teins, with the transformed lysozyme (i.e. a-lactalbumin)
serving to alter sugar synthesis such that new milk-speciﬁc
sugars were formed. Over a period of perhaps 150 million
years, and during the course of multiple radiations of early
synapsids and the descendent lineages leading to mammals,
this secretory ﬂuid and the glands that produced it became
more complex, the volumes produced became greater, and
the extent of dependence by hatchlings and growing young
increased. The presence of a glandular skin secretion
appears to have been essential to endothermic incubation of
eggs with parchment-like shells. The sequential radiations of
basal synapsids, therapsids, cynodonts, and mammalialforms
became increasingly mammal-like in terms of metabolism,
locomotor ability, structural adaptations to diet, and repro-
duction. On the one hand, the predominance of milk secretion
led to reduction in egg yolk mass and smaller and less-devel-
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proteins, and relatively high levels of lactose. Perhaps such
species were selected because their milks bore resemblance
to human milk, in being dilute, or perhaps such species were
easier to milk because their dilute milks accumulated in
substantial storage cisterns between bouts of suckling.
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