ArticlePDF AvailableLiterature Review

The evolution of milk secretion and its ancient origins



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 α-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 β-lactoglobulin and whey acidic protein, which with α-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 170 mya) 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.
(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
into being.
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
et al
2010); terrestrial-breeding frogs provide water, antimicrobial
compounds, and perhaps some nutrients to their eggs via
granular and/or mucous gland secretions (Taigen
et al
., 1984;
Oftedal, 2002a); and the young of some live-bearing caecilians
feed on sloughed skin, skin secretions, or perhaps both
et al
., 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
et al
., 2007), but may be as little as
3–4 days in the ice-breeding hooded seal (Oftedal
et al
., 1993a).
Lactation is highly complex and apparently of ancient evo-
lutionary origin (Oftedal, 2002b; Lefevre
et al
., 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
et al
., 2009).
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
et al
., 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
et al
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
et al
., 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
et al
., 2006; McClellan
et al
., 2008))
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
et al
., 2007). Some lizards and snakes overcome this
constraint by internal egg-retention, including development of
placental structures to provide nutrients to the developing
embryos (Thompson
et al
., 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
et al
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
et al
., 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
in utero
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
(Oftedal, 2002b).
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
(Dhouailly, 2009).
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
et al
. (2006) speculated that mammary secretion first
developed as part of an inflammatory response by mucous
secreting cells, and McClellan
et al
. (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
et al
., 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
et al
. (2006) are certainly correct that mammary
glands must derive, ultimately, from the simple glandular
skin structures found in pre-amniotic tetrapods (Quagliata
et al
., 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
et al
., 2007).
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
et al
., 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
et al
., 2008; Mayer
et al
., 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
et al
. (2008) reduced BMP
signaling in the mouse by transgenic overexpression of a
BMP antagonist, nipple epithelium was converted into pilo-
sebaceous units. Mayer
et al
. (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
et al
. (2006)
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
et al
., 1999; Fujita, 2002),
and thus the co-option of innate immune system components
into the regulatory elements of epithelial–mesenchyme sig-
naling (McClellan
et al
., 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
understood (Smolenski
et al
., 2007; Lemay
et al
., 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
et al
., 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
et al
., 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
et al
., 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
et al
2011). On the basis of the relative locations and structures of
exons of these P/Q-rich SCPPs, as well as their phylogenetic
distribution, Kawasaki
et al
. (2011) proposed that the a- and
b-caseins derived via gene duplication and exon changes
from an ancestral gene (
) that derives from another
gene, either
(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;
et al
., 2011). Kawasaki
et al
. (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
eggshell. Kawasaki
et al
. (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
et al
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
et al
., 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
et al
., 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
et al
., 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
masses (Vorbach
et al
., 2002; Ogg
et al
., 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
genes (
) that code for the family of proteins known as
butyrophilins (Rhodes
et al
., 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
et al
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
in vitro
et al
., 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
et al
., 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
et al
., 2003). The
MFEs are believed to have evolved as an ancestral XOR in
prokaryotes (Garattini
et al
., 2003). Although the XOR gene is
sometimes considered to code for a housekeeping protein
et al
., 2002), this is debatable, as XOR is unequally
expressed in cells (Garattini
et al
., 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
et al
., 2000; Nishino
et al
., 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
et al
., 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
et al
., 2002); the binding of XOR to the B30.2
domain of butyrophilin (Jeong
et al
., 2009); and the failure of
MFG formation in heterozygous XOR knockout mice (Vorbach
et al
., 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
et al
., 1997;
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;
et al
., 2003; Stoeckelhuber
et al
., 2006;
et al
., 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
yolk (Brawand
et al
., 2008).
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
et al
., 2001; Messer and Urashima, 2002; Uemura
et al
., 2009;
et al
., 2010). Both lactose and oligosaccharides with
lactose at the reducing end are unique to milk (Toba
et al
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
(Brew, 2003).
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
et al
., 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
per se
. 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
et al
., 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
et al
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
et al
., 2005; Reich and Arnould, 2007; Sharp
et al
., 2008);
therefore the milk is devoid of lactose or lactose-based
oligosaccharides (Oftedal
et al
., 1987a and 2011). These taxa
manage to produce large volumes of high-fat milk (Oftedal
et al
., 1987b; Arnould and Boyd, 1995; Arnould
et al
., 1996),
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
et al
., 1994; Stacey
et al
., 1995).
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
et al
., 1977; Burns and
Milner, 1981; Fuller
et al
., 1989; National Research Council,
1995). Methionine can substitute for cysteine in most cases
(except, perhaps, in premature human infants; Fomon
et al
1986; Thomas
et al
., 2008), but cysteine can only replace
approximately half of the methionine requirement in growing
animals (Fuller
et al
., 1989). In formulating casein-based diets,
supplemental cysteine or methionine are required to compen-
et al
., 1993;
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
et al
., 1987; Piotte and Grigor, 1996; Demmer
et al
., 1998;
et al
., 1998).
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
et al
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
et al
. (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,
1996; Akerstrom
et al
., 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
end (Ganfornina
et al
., 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
amphibia (Ganfornina
et al
., 2000; Sanchez
et al
., 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
et al
., 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
et al
., 2004). Other lipocalins (trichosurin, late lactation
protein) are expressed in marsupial milk (Demmer
et al
., 1998;
et al
., 1998); however, these are only distantly related
to b-lactoglobulin and are apparently of more recent origin
et al
., 2000).
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,
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
et al
., 2003; Bingle
et al
., 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
et al
., 2006; Sharp
et al
., 2007).
There is evidence that WFDC domains influence cell pro-
liferation and growth
in vitro
and in transgenic mice
(reviewed by Topcic
et al
., 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
et al
., 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
et al
., 2006), and in antimicrobial response as part of the
innate immunity of crustaceans and perhaps insects (Zou
et al
., 2007; Jia
et al
., 2008; Smith
et al
., 2010b). A number
of WFDC domain-containing proteins are also secreted by
snake venom glands, where they have antibacterial function
et al
., 2007; Fry
et al
., 2008), and by skin glands in
frogs, where they serve as antimicrobial defensive com-
pounds (Ali
et al
., 2002; Zhang
et al
., 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
et al
2003; Bingle and Vyakarnam, 2008), and in frog skin secre-
tions (Ali
et al
., 2002; Zhang
et al
., 2009).
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
et al
., 1999). Monotreme and marsupial WAPs contain two
to three WFDC domains, but of differing sequence and
arrangement than eutherian WAPs (Sharp
et al
., 2007), and
it is thought that they may retain functions lost in eutherians,
but more evidence is required (Topcic
et al
., 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
et al
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
et al
., 2006; Brawand
et al
., 2008; McClellan
et al
., 2008;
Capuco and Akers, 2009; Lemay
et al
., 2009; Lefevre
et al
2010; Kawasaki
et al
., 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-
oped hatchlings; on the other hand, growth patterns changed
as offspring relied for longer periods on milk, with delayed
tooth eruption and more specialized mature dentition. It was
only because lactation was far advanced that Triassic and Jur-
assic mammaliaforms could become so tiny, with adult body
masses of some taxa weighing only a few grams.
Despite the tremendous diversity in milk composition among
extant mammals, the specific constituents of milk are common
to the primary mammalian lineages, reflecting that they were
inherited from pre-mammalian taxa. The major qualitative
differences that have arisen among mammalian taxa (such as
lack of a-lactalbumin in fur seals and sea lions, lack of WAP in
ruminants, and lack of b-lactoglobulin in humans) are due
to losses of functional components, rather than
de novo
tion of new constituents. The evolution of the placenta in
eutherians also removed much of the need for nutrient transfer
during early development. Marsupials, which have no or
very simple placentas, rely on lactation for a greater span of
development than eutherians, and not surprisingly, marsupial
milks undergo much greater changes in composition over the
course of lactation than do most eutherian milks (Oftedal and
Iverson, 1995). It is not known whether some of the unique
whey proteins that have been found in marsupials represent
milk constituents that were lost during eutherian evolution, or
if they have evolved since marsupials and eutherians diverged.
When human civilizations finally developed animal agri-
culture, they were able to select as dairy animals a suite
of species (e.g. cows, sheep, goats, water buffaloes, yaks,
camels, horses, and asses) characterized by relatively dilute
milks containing modest levels of fat, caseins, and whey
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.
However, it is only now that we are beginning to appreciate
that the remarkable secretory product that is the foundation
of the dairy industry had such a very long evolutionary history
before mammals appeared on earth.
Akerstrom B, Borregaard N, Flower DR and Salier J-P 2006. Lipocalins. Landes
Bioscience, Georgetown, TX.
Ali MF, Lips KR, Knoop FC, Fritzsch B, Miller C and Conlon JM 2002.
Antimicrobial peptides and protease inhibitors in the skin secretions of the
crawfish frog,
Rana areolata
. Biochimica et Biophysica Acta (BBA) – Proteins
and Proteomics 1601, 55–63.
Andrechek ER, Mori S, Rempel RE, Chang JTand Nevins JR 2008. Patterns of cell
signaling pathway activation that characterize mammary development.
Development 135, 2403–2413.
Arnould JPY and Boyd IL 1995. Temporal patterns of milk production in Antarctic
fur seals (
Arctocephalus gazella
). Journal of Zoology 237, 1–12.
Arnould JPY, Boyd IL and Socha DG 1996. Milk consumption and growth
efficiency in Antarctic fur seal (
Arctocephalus gazella
) pups. Canadian Journal of
Zoology 74, 254–266.
Beck G and Habicht GS 1996. Immunity and the invertebrates. Scientific
American 275, 60–63, 66.
Beutler B 2004. Innate immunity: an overview. Molecular Immunology 40, 845–859.
Bingle CD and Vyakarnam A 2008. Novel innate immune functions of the whey
acidic protein family. Trends in Immunology 29, 444–453.
Bingle L, Cross SS, High AS, Wallace WA, Rassl D, Yuan G, Hellstrom I, Campos
MA and Bingle CD 2006. WFDC2 (HE4): a potential role in the innate immunity
of the oral cavity and respiratory tract and the development of adenocarcinomas
of the lung. Respiratory Research 7, 61–70.
Blackburn DG 2006. Squamate reptiles as model organisms for the evolution of
viviparity. Herpetological Monographs 20, 131–146.
Brawand D, Wahli Wand Kaessmann H 2008. Loss of egg yolk genes in mammals
and the origin of lactation and placentation. PLoS Biology 6, e63. doi:10.1371/
Bresslau E 1912. Die entwickelung des mammarapparates der monotremen,
marsupialier und einiger placentallier. III. Entwickelung des mammarapparates
der marsupialier, insectivoren, nagatheire, carnivoren und widerkauer. Jenaische
Denkschriften 7, 647–874, plates 637–646.
Bresslau E 1920. The mammary apparatus of the mammals in light of
ontogenesis and phylogenesis. Methuen, London, UK.
Brew K 2003. a-lactalbumin. In Advanced Dairy Chemistry – I. Proteins. Part A
(ed. PF Fox and P McSweeney), pp. 387–419. Kluver Academic, New York, NY.
Buckley J, Maunder RJ, Foey A, Pearce J, Val AL and Sloman KA 2010. Biparental
mucus feeding: a unique example of parental care in an Amazonian cichlid. The
Journal of Experimental Biology 213, 3787–3795.
Burns RA and Milner JA 1981. Sulfur amino acid requirements of immature
Beagle dogs. The Journal of Nutrition 111, 2117–2124.
Callewaert L and Michiels CW 2010. Lysozymes in the animal kingdom. Journal
of Biosciences 35, 127–160.
Capuco AV and Akers RM 2009. Minireview. The origin and evolution of
lactation. Journal of Biology 8, 37. doi:10.1186/jbiol139
Chudinov P 1968. Structure of the integuments of thermomorphs. Doklady
Academy of Sciences USSR Earth Science Section 179, 226–229.
Clarke B 1997. The natural history of amphibian skin secretions, their normal
functioning and potential medical applications. Biological Reviews 72, 365–379.
Crisp EA, Messer M and Cowan PE 1989. Intestinal lactase (b-galactosidase)
and other disaccharidase activities of suckling and adult common brushtail
Trichosurus vulpecula
(Marsupialia: Phalangeridae). Reproduction,
Fertility, and Development 1, 315–324.
Darwin C 1872. On the origin of species by means of natural selection, or the
preservation of favoured races in the struggle of life. John Murray, London, UK.
Demmer J, Ross IK, Ginger MR, Piotte CK and Grigor MR 1998. Differential
expression of milk protein genes during lactation in the common brushtail
possum (
Trichosurus vulpecula
). Journal of Molecular Endocrinology 20, 37–44.
Dhouailly D 2009. A new scenario for the evolutionary origin of hair, feather, and
avian scales. Journal of Anatomy 214, 587–606.
Enroth C, Eger BT, Okamoto K, Nishino T and Pai EF 2000. Crystal structures of
bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based
mechanism of conversion. Proceedings of the National Academy of Sciences of
the United States of America 97, 10723–10728.
Flower DR 1996. The lipocalin protein family: structure and function. The
Biochemical Journal 318, 1–14.
Foldager J, Huber JT and Bergen WG 1977. Methionine and sulfur amino acid
requirement in the preruminant calf. Journal of Dairy Science 60, 1095–1104.
Fomon SJ, Ziegler EE, Nelson SE and Edwards BB 1986. Requirement for sulfur-
containing amino acids in infancy. The Journal of Nutrition 116, 1405–1422.
Fox PF 2003. Milk proteins: general and historical aspects. In Advanced dairy
chemistry – I. Proteins. Part A (ed. PF Fox and P McSweeney), pp. 1–48. Kluwer
Academic, New York, NY.
Evolution of milk secretion
Fry BG, Scheib H, van der Weerd L, Young B, McNaughtan J, Ramjan SFR, Vidal
N, Poelmann RE and Norman JA 2008. Evolution of an arsenal: structural and
functional diversification of the venom system in the advanced snakes
(Caenophidia). Molecular & Cellular Proteomics 7, 215–246.
Fujita T 2002. Evolution of the lectin–complement pathway and its role in innate
immunity. Nature Reviews Immunology 2, 346–353.
Fuller MF, McWilliam R, Wang TC and Giles LR 1989. The optimum dietary
amino acid pattern for growing pigs. 2. Requirements for maintenance and for
tissue protein accretion. The British Journal of Nutrition 62, 255–267.
Ganfornina MD, Gutierrez G, Bastiani M and Sanchez D 2000. A phylogenetic
analysis of the lipocalin protein family. Molecular Biology and Evolution 17,
Ganfornina MD, Sanchez D, Greene LH and Flower DR 2006. The Lipocalin
protein family: protein sequence, structure and relationship to the calycin
superfamily. In Lipocalins (ed. B Akerstrom, N Borregaard, DR Flower and
J-P Salier), pp. 17–27. Landes Bioscience, Georgetown, TX.
Garattini E, Mendel R, RomA
˜£o MJ, Wright R and Terao M 2003. Mammalian
molybdo-flavoenzymes, an expanding family of proteins: structure, genetics,
regulation, function and pathophysiology. Biochemical Journal 372, 15–32.
Gesase AP and Satoh Y 2003. Apocrine secretory mechanism: recent findings
and unresolved problems. Histology and Histopathology 18, 597–608.
Gregory WK 1910. The orders of mammals. Bulletin of the American Museum of
Natural History 27, 1–524.
Griffiths M 1978. Biology of the monotremes. Academic Press, New York, NY.
Gritli-Linde A, Hallberg K, Harfe BD, Reyahi A, Kannius-Janson M, Nilsson J,
Cobourne MT, Sharpe PT, McMahon AP and Linde A 2007. Abnormal hair
development and apparent follicular transformation to mammary gland in the
absence of hedgehog signaling. Developmental Cell 12, 99–112.
Hagiwara K, Kikuchi T, Endo Y, Huqun, Usui K, Takahashi M, Shibata N,
Kusakabe T, Xin H, Hoshi S, Miki M, Inooka N, Tokue Y and Nukiwa T 2003.
Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single
whey acidic protein motif. Journal of Immunology 170, 1973–1979.
Hajjoubi S, Rival-Gervier S, Hayes H, Floriot S, Eggen A, Piumi F, Chardon P,
Houdebine LM and Thepot D 2006. Ruminants genome no longer contains whey
acidic protein gene but only a pseudogene. Gene 370, 104–112.
Hatsell SJ and Cowin P 2006. Gli3-mediated repression of Hedgehog targets is
required for normal mammary development. Development 133, 3661–3670.
Hayssen V and Blackburn DG 1985. a-lactalbumin and the origins of lactation.
Evolution 39, 1147–1149.
Hiemstra PS 2002. Novel roles of protease inhibitors in infection and
inflammation. Biochemical Society Transactions 30, 116–120.
Hoffmann JA, Kafatos FC, Janeway CA and Ezekowitz RA 1999. Phylogenetic
perspectives in innate immunity. Science 284, 1313–1318.
Hood WR, Oftedal OT and Kunz TH 2011. Is tissue maturation necessary for
flight? Changes in body composition during postnatal development in the big
brown bat. Journal of Comparative Physiology B: Biochemical, Systemic, and
Environmental Physiology 181, 423–435.
Hopson JA 1973. Endothermy, small size and the origin of mammalian
reproduction. American Naturalist 107, 446–452.
Horseman ND and Buntin JD 1995. Regulation of pigeon cropmilk secretion and
parental behaviors by prolactin. Annual Review of Nutrition 15, 213–238.
Jenssen H, Hamill P and Hancock REW 2006. Peptide antimicrobial agents.
Clinical Microbiology Reviews 19, 491–511.
Jeong J, Rao AU, Xu J, Ogg SL, Hathout Y, Fenselau C and Mather IH 2009. The
PRY/SPRY/B30. 2 Domain of butyrophilin 1A1 (BTN1A1) binds to xanthine
oxidoreductase. Journal of Biological Chemistry 284, 22444–22456.
Jia Y, Sun Y, Wang Z, Wang Q, Wang X, Zhao X and Wang J 2008. A single whey
acidic protein domain (SWD)-containing peptide from fleshy prawn with
antimicrobial and proteinase inhibitory activities. Aquaculture 284, 246–259.
Kawasaki K 2009. The SCPP gene repertoire in bony vertebrates and graded
differences in mineralized tissues. Development Genes and Evolution 219, 147–157.
Kawasaki K and Weiss KM 2003. Mineralized tissue and vertebrate evolution:
the secretory calcium-binding phosphoprotein gene cluster. Proceedings of the
National Academy of Sciences of the United States of America 100, 4060–4065.
Kawasaki K, Lafont A and Sire J 2011. The evolution of milk casein genes from
tooth genes before the origin of mammals. Molecular Biology and Evolution 28,
Kemp TS 2005. The origin and evolution of mammals. Oxford University Press,
New York, NY.
Kontopidis G, Holt C and Sawyer L 2004. Invited review: beta-lactoglobulin:
binding properties, structure, and function. Journal of Dairy Science 87,
Konuma T, Sakurai K and Goto Y 2007. Promiscuous binding of ligands by
b-lactoglobulin involves hydrophobic interactions and plasticity. Journal of
Molecular Biology 368, 209–218.
Kupfer A, Mu
¨ller H, Antoniazzi MM, Jared C, Greven H, Nussbaum RA and
Wilkinson M 2006. Parental investment by skin feeding in a caecilian
amphibian. Nature 440, 926–929.
Lefevre CM, Sharp JA and Nicholas KR 2009. Characterisation of monotreme
caseins reveals lineage-specific expansion of an ancestral casein locus in
mammals. Reproduction, Fertility, and Development 21, 1015–1027.
Lefevre CM, Sharp JA and Nicholas KR 2010. Evolution of lactation: ancient
origin and extreme adaptations of the lactation system. Annual Review of
Genomics and Human Genetics 11, 219–238.
Lemay DG, LynnDJ, Martin WF, Neville MC, Casey TM, Rincon G, Kriventseva EV,
Barris WC, Hinrichs AS, Molenaar AJ, Pollard KS, Maqbool NJ, Singh K, Murney
R, Zdobnov EM, Tellam RL, Medrano JF, German JB and Rijnkels M 2009. The
bovine lactation genome: insights into the evolution of mammalian milk.
Genome Biology 10, R43. doi:10.1186/gb-2009-10-4-r43.
Lillywhite HB 2006. Water relations of tetrapod integument. The Journal of
Experimental Biology 209, 202–226.
Lillywhite HB, Mittal AK, Garg TK and Agrawal N 1997. Integumentary structure
and its relationship to wiping behaviour in the common Indian tree frog,
Polypedates maculatus
. Journal of Zoology 243, 675–687.
Lourdais O, Hoffman TCM and Denardo DF 2007. Maternal brooding in the
children’s python (
Antaresia childreni
) promotes egg water balance. Journal of
Comparative physiology B: Biochemical, Systemic, and Environmental Physiol-
ogy 177, 569–577.
Lowe JB and Varki A 1999. Glycosyltransferases. In Essentials of glycobiology
(ed. A Varki, R Cummings, J Esko, H Freeze, G Hart and J Marth), pp. 253–266.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Luo ZX, Crompton AW and Sun AL 2001. A new mammaliaform from the early
Jurassic and evolution of mammalian characteristics. Science 292, 1535–1540.
Martin HM, Hancock JT, Salisbury V and Harrison R 2004. Role of xanthine
oxidoreductase as an antimicrobial agent. Infection and Immunity 72, 4933–4939.
Mather IH 2011. Milk lipids. Milk fat globule membrane. In Encyclopedia of dairy
sciences (ed. J Fuquay, PF Fox and P McSweeney), vol. 3, 2nd edition, pp. 680–690.
Academic Press, San Diego.
Mather IH and Keenan TW 1998. Origin and secretion of milk lipids. Journal of
Mammary Gland Biology and Neoplasia 3, 259–273.
Mayer JA, Foley J, De La Cruz D, Chuong C-M and Widelitz R 2008. Conversion
of the nipple to hair-bearing epithelia by lowering bone morphogenetic protein
pathway activity at the dermal–epidermal interface. The American Journal of
Pathology 173, 1339–1348.
McClellan HL, Miller SJ and Hartmann PE 2008. Evolution of lactation: nutrition
v. protection with special reference to five mammalian species. Nutrition
Research Reviews 21, 97–116.
McManaman JL, Reyland ME and Thrower EC 2006. Secretion and fluid
transport mechanisms in the mammary gland: comparisons with the exocrine
pancreas and the salivary gland. Journal of Mammary Gland Biology and
Neoplasia 11, 249–268.
McManaman JL, Palmer CA, Wright RM and Neville MC 2002. Functional
regulation of xanthine oxidoreductase expression and localization in the mouse
mammary gland: evidence of a role in lipid secretion. The Journal of Physiology
545, 567–579.
Messer M and Urashima T 2002. Evolution of milk oligosaccharides and lactose.
Trends in Glycoscience and Glycotechnology 14, 153–176.
Nair DG, Fry BG, Alewood P, Kumar PP and Kini RM 2007. Antimicrobial activity
of omwaprin, a new member of the waprin family of snake venom proteins. The
Biochemical Journal 402, 93–104.
National Research Council 1995. Nutrient requirements of laboratory animals.
National Academy Press, Washington, DC.
Nelson CM and Bissell MJ 2006. Of extracellular matrix, scaffolds, and
signaling: tissue architecture regulates development, homeostasis, and cancer.
Annual Review of Cell and Developmental Biology 22, 287–309.
Newburg DS 1996. Oligosaccharides and glycoconjugates in human milk: their
role in host defense. Journal of Mammary Gland Biology and Neoplasia 1,
Nicholas KR, Messer M, Elliott C, Maher F and Shaw DC 1987. A novel whey
protein synthesized only in late lactation by the mammary gland from the
tammar (
Macropus eugenii
). The Biochemical Journal 241, 899–904.
Nishino T, Okamoto K, Eger BT, Pai EFand Nishino T 2008. Mammalian xanthine
oxidoreductase – mechanism of transition from xanthine dehydrogenase to
xanthine oxidase. The FEBS Journal 275, 3278–3289.
Novacek MJ, Rougier GW, Wible JR, McKenna MC, Dashzeveg D and Horovitz I
1997. Epipubic bones in eutherian mammals from the Late Cretaceous of
Mongolia. Nature 389, 483–486.
Oftedal OT 1997. Lactation in whales and dolphins: evidence of divergence
between baleen- and toothed-species. Journal of Mammary Gland Biology and
Neoplasia 2, 205–230.
Oftedal OT 2000. Use of maternal reserves as a lactation strategy in large
mammals. Proceedings of the Nutrition Society 59, 99–106.
Oftedal OT 2002a. The origin of lactation as a water source for parchment-
shelled eggs. Journal of Mammary Gland Biology and Neoplasia 7, 253–266.
Oftedal OT 2002b. The mammary gland and its origin during synapsid evolution.
Journal of Mammary Gland Biology and Neoplasia 7, 225–252.
Oftedal OT 2011. Milk of marine mammals. In Encyclopedia of dairy sciences
(ed. J Fuquay, PF Fox and P McSweeney), vol. 3, 2nd edition, pp. 563–580.
Academic Press, San Diego, CA.
Oftedal OT and Gittleman JL 1989. Patterns of energy output during
reproduction in carnivores. In Carnivore behavior, ecology and evolution
(ed. JL Gittleman), pp. 355–378. Cornell University Press, Ithaca, NY.
Oftedal OT and Iverson SJ 1995. Comparative analysis of non-human milks. A.
phylogenetic variation in the gross composition of milks. In Handbook of milk
composition (ed. RG Jensen), pp. 749–789. Academic Press, San Diego, CA.
Oftedal OT, Boness DJ and Tedman RA 1987a. The behavior, physiology, and
anatomy of lactation in the Pinnipedia. Current Mammalogy 1, 175–245.
Oftedal OT, Iverson SJ and Boness DJ 1987b. Milk and energy intakes of
suckling California sea lion
Zalophus californianus
pups in relation to sex,
growth, and predicted maintenance requirements. Physiological Zoology 60,
Oftedal OT, Bowen DW and Boness DJ 1993a. Energy transfer by lactating
hooded seals and nutrient deposition in their pups during the 4 days from birth
to weaning. Physiological Zoology 66, 412–436.
Oftedal OT, Alt GL, Widdowson EM and Jakubasz MR 1993b. Nutrition and
growth of suckling black bears (
Ursus americanus
) during their mothers’ winter
fast. British Journal of Nutrition 70, 59–79.
Ogg SL, Weldon AK, Dobbie L, Smith AJH and Mather IH 2004. Expression of
butyrophilin (Btn1a1) in lactating mammary gland is essential for the regulated
secretion of milk-lipid droplets. Proceedings of the National Academy of
Sciences of the United States of America 101, 10084–10089.
Packard MJ and Seymour RS 1997. Evolution of the amniote egg. In Amniote
origins: completing the transition to land (ed. SS Sumida and KLM Martin),
pp. 265–290. Academic Press, San Diego, CA.
Perez MD and Calvo M 1995. Interaction of beta-lactoglobulin with retinol and
fatty acids and its role as a possible biological function for this protein: a review.
Journal of Dairy Science 78, 978–988.
Pervaiz S and Brew K 1985. Homology of b-lactoglobulin, serum retinol-binding
protein, and protein HC. Science 228, 335–337.
Piotte CP and Grigor MR 1996. A novel marsupial protein expressed by the
mammary gland only during the early lactation and related to the Kunitz
proteinase inhibitors. Archives of Biochemistry and Biophysics 330, 59–64.
Piotte CP, Hunter AK, Marshall CJ and Grigor MR 1998. Phylogenetic analysis of
three lipocalin-like proteins present in the milk of
Trichosurus vulpecula
(Phalangeridae, Marsupialia). Journal of Molecular Evolution 46, 361–369.
Pond CM 1977. The significance of lactation in the evolution of mammals.
Evolution 31, 177–199.
Prager EM and Wilson AC 1988. Ancient origin of lactalbumin from lysozyme:
analysis of DNA and amino acid sequences. Journal of Molecular Evolution 27,
Qasba PK and Kumar S 1997. Molecular divergence of lysozymes and
a-lactalbumin. Critical Reviews in Biochemistry and Molecular Biology 32,
Quagliata S, Malentacchi C, Delfino C, Brunasso AMG and Delfino G 2006.
Adaptive evolution of secretory cell lines in vertebrate skin. Caryologia 59,
Ramakrishnan B and Qasba PK 2001. Crystal structure of lactose synthase
reveals a large conformational change in its catalytic component, the b-1,4-
galactosyltransferase-I. Journal of Molecular Biology 310, 205–218.
Ranganathan S, Simpson KJ, Shaw DC and Nicholas KR 1999. The whey acidic
protein family: a new signature motif and three-dimensional structure by
comparative modeling. Journal of Molecular Graphics & Modelling 17,
106–113, 134–136.
Reeves PG, Nielsen FH and Fahey GC 1993. AIN-93 purified diets for laboratory
rodents: final report of the American Institute of Nutrition ad hoc writing
committee on the reformulation of the AIN-76A rodent diet. The Journal of
Nutrition 123, 1939–1951.
Reich C and Arnould J 2007. Evolution of Pinnipedia lactation strategies: a
potential role for a-lactalbumin? Biology Letters 3, 546–549.
Reilly SM and White TD 2003. Hypaxial motor patterns and the function of
epipubic bones in primitive mammals. Science 299, 400–402.
Rhodes DA, Stammers M, Malcherek G, Beck S and Trowsdale J 2001. The
cluster of BTN genes in the extended major histocompatibility complex.
Genomics 71, 351–362.
Rijnkels M 2002. Multispecies comparison of the casein gene loci and evolution
of casein gene family. Journal of Mammary Gland Biology and Neoplasia 7,
Rijnkels M 2003. Multispecies comparative analysis of a mammalian-specific
genomic domain encoding secretory proteins. Genomics 82, 417–432.
Robinson GW 2004. Identification of signaling pathways in early mammary
gland development by mouse genetics. Breast Cancer Research 6, 105–108.
Sanchez D, Ganfornina MD, Gutierrez G and Marin A 2003. Exon–intron
structure and evolution of the Lipocalin gene family. Molecular Biology and
Evolution 20, 775–783.
Sanchez D, Ganfornina MD, Gutierrez G, Gauthier-Jauneau A-C, Risler J-L and
Salier J-P 2006. Lipocalin genes and their evolutionary history. In Lipocalins
(ed. B Akerstrom, N Borregaard, DR Flower and J-P Salier), pp. 5–16. Landes
Bioscience, Georgetown, TX.
Sawyer L 2003. b-lactoglobulin. In Advanced dairy chemistry – I. Proteins. Part A
(ed. PF Fox and P McSweeney), pp. 319–386. Kluwer Academic, New York, NY.
Senda A, Hatakeyama E, Kobayashi R, Fukuda K, Uemura Y, Saito T, Packer C,
Oftedal OT and Urashima T 2010. Chemical characterization of milk
oligosaccharides of an African lion (
Panthera leo
) and a clouded leopard
Neofelis nebulosa
). Animal Science Journal 81, 687–693.
Shaper NL, Charron M, Lo NW and Shaper JH 1998. b-1,4-galactosyltransferase
and lactose biosynthesis: recruitment of a housekeeping gene from the
nonmammalian vertebrate gene pool for a mammary gland specific function.
Journal of Mammary Gland Biology and Neoplasia 3, 315–324.
Sharp JA, Lefevre C and Nicholas KR 2007. Molecular evolution of monotreme
and marsupial whey acidic protein genes. Evolution & Development 9, 378–392.
Sharp JA, Lefevre C and Nicholas KR 2008. Lack of functional alpha-lactalbumin
prevents involution in Cape fur seals and identifies the protein as an apoptotic
milk factor in mammary gland involution. BMC Biology 6, 48. doi:10.1186/
Sharp JA, Cane KN, Lefevre C, Arnould JPY and Nicholas KR 2005. Fur seal
adaptations to lactation: insights into mammary gland function. Current Topics
in Developmental Biology 72, 275–308.
Shennan DB and Peaker M 2000. Transport of milk constituents by the
mammary gland. Physiological Reviews 80, 925–951.
Sidor CA and Hopson JA 1998. Ghost lineages and ‘‘mammalness’’: assessing
the temporal pattern of character acquisition in the Synapsida. Paleobiology 24,
Smith IA, Knezevic BR, Ammann JU, Rhodes DA, Aw D, Palmer DB, Mather IH and
Trowsdale J 2010a. BTN1A1, the mammary gland butyrophilin, and BTN2A2 are
both inhibitors of T cell activation. The Journal of Immunology 184, 3514–3525.
Smith VJ, Desbois AP and Dyrynda EA 2010b. Conventional and unconventional
antimicrobials from fish, marine invertebrates and micro-algae. Marine Drugs 8,
Smolenski G, Haines S, Fiona YSK, Bond J, Farr V, Davis SR, Stelwagen K and
Wheeler TT 2007. Characterisation of host defence proteins in milk using a
proteomic approach. Journal of Proteome Research 6, 207–215.
Evolution of milk secretion
Smyth E, Clegg RA and Holt C 2004. A biological perspective on the structure
and function of caseins and casein micelles. International Journal of Dairy
Technology 57, 121–126.
Stacey A, Schnieke A, Kerr M, Scott A, McKee C, Cottingham I, Binas B, Wilde C
and Colman A 1995. Lactation is disrupted by a-lactalbumin deficiency and can
be restored by human a-lactalbumin gene replacement in mice. Proceedings of
the National Academy of Sciences of the United States of America 92,
Starck JM and Ricklefs R 1998. Avian growth and development: evolution
within the altricial-precocial spectrum. Oxford University Press, New York, NY.
Stinnakre MG, Vilotte JL, Soulier S and Mercier JC 1994. Creation and
phenotypic analysis of alpha-lactalbumin-deficient mice. Proceedings of the
National Academy of Sciences of the United States of America 91, 6544–6548.
Stoeckelhuber M, Stoeckelhuber BM and Welsch U 2003. Human glands of Moll:
histochemical and ultrastructural characterization of the glands of Moll in the
human eyelid. Journal of Investigative Dermatology 121, 28–36.
Stoeckelhuber M, Schubert C, Kesting MR, Loeffelbein DJ, Nieberler M, Koehler
C and Welsch U 2011. Human axillary apocrine glands: proteins involved in the
apocrine secretory mechanism. Histology and Histopathology 26, 177–184.
Stoeckelhuber M, Matthias C, Andratschke M, Stoeckelhuber BM, Koehler C,
Herzmann S, Sulz A and Welsch U 2006. Human ceruminous gland:
ultrastructure and histochemical analysis of antimicrobial and cytoskeletal
components. The Anatomical Record Part A: Discoveries in Molecular, Cellular,
and Evolutionary Biology 288A, 877–884.
Taigen TL, Pough FH and Stewart MM 1984. Water balance of terrestrial anuran
Eleutherodactylus coqui
) eggs: importance of parental care. Ecology 65, 248–255.
Thomas B, Gruca LL, Bennett C and Parimi PS 2008. Metabolism of methionine
in the newborn infant: response to the parenteral and enteral administration of
nutrients. Pediatric Research 64, 381–386.
Thompson MB, Biazik JB, Lui S, Adams SM and Murphy CR 2006. Morphological
and functional changes to the uterus of lizards with different placental
complexities. Herpetological Monographs 20, 178–185.
Toba T, Nagashima S and Adachi S 1991. Is lactose really present in plants?
Journal of the Science of Food and Agriculture 54, 305–308.
Topcic D, Auguste A, De Leo AA, Lefevre C, Digby MR and Nicholas KR 2009.
Characterization of the tammar wallaby (
Macropus eugenii
) whey acidic protein
gene; new insights into the function of the protein. Evolution & Development
11, 363–375.
Treccani L, Mann K, Heinemann Fand Fritz M 2006. Perlwapin, an abalone nacre
protein with three four-disulfide core (whey acidic protein) domains, inhibits the
growth of calcium carbonate crystals. Biophysical Journal 91, 2601–2608.
Triplett AA, Sakamoto K, Matulka LA, Shen L, Smith GH and Wagner KU 2005.
Expression of the whey acidic protein (Wap) is necessary for adequate
nourishment of the offspring but not functional differentiation of mammary
epithelial cells. Genesis 43, 1–11.
Tyndale-Biscoe H and Renfree M 1987. Reproductive physiology of marsupials.
Cambridge University Press, Cambridge, UK.
Uemura Y, Takahashi S, Senda A, Fukuda K, Saito T, Oftedal OT and Urashima T
2009. Chemical characterization of milk oligosaccharides of a spotted hyena
Crocuta crocuta
). Comparative Biochemistry and Physiology A: Molecular &
Integrative Physiology 152, 158–161.
Urashima T, Saito T, Nakamura T and Messer M 2001. Oligosaccharides of milk
and colostrum in non-human mammals. Glycoconjugate Journal 18, 357–371.
Varki A 1998. Factors controlling the glycosylation potential of the Golgi
apparatus. Trends in Cell Biology 8, 34–40.
Vorbach C 2003. Xanthine oxidoreductase is central to the evolution and
function of the innate immune system. Trends in Immunology 24, 512–517.
Vorbach C, Scriven A and Capecchi MR 2002. The housekeeping gene xanthine
oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene
sharing in the lactating mammary gland. Genes & Development 16, 3223–3235.
Vorbach C, Capecchi MR and Penninger JM 2006. Evolution of the mammary
gland from the innate immune system? BioEssays: News and Reviews in
Molecular, Cellular and Developmental Biology 28, 606–616.
Watson CJ and Khaled WT 2008. Mammary development in the embryo
and adult: a journey of morphogenesis and commitment. Development 135,
West KL, Oftedal OT, Carpenter JR, Krames BJ, Campbell M and Sweeney JC
2007. Effect of lactation stage and concurrent pregnancy on milk composition in
the bottlenose dolphin. Journal of Zoology 273, 148–160.
Yang MC, Chen NC, Chen C-J, Wu CY and Mao SJT 2009. Evidence for beta-
lactoglobulin involvement in vitamin D transport in vivo – role of the gamma-
turn (Leu-Pro-Met) of beta-lactoglobulin in vitamin D binding. FEBS Journal 276,
Zhang Z, Zhang B, Nie X, Liu Q, Xie F and Shang D 2009. Transcriptome analysis
and identification of genes related to immune function in skin of the Chinese
brown frog. Zoological Science 26, 80–86.
Zhao Y, Jin Y, Lee W and Zhang Y 2006. Purification of a lysozyme from skin
secretions of
Bufo andrewsi
. Comparative Biochemistry and Physiology C:
Toxicology & Pharmacology 142, 46–52.
Zou Z, Evans JD, Lu Z, Zhao P, Williams M, Sumathipala N, Hetru C, Hultmark D
and Jiang H 2007. Comparative genomic analysis of the
system. Genome Biology 8, R177. doi:10.1186/gb-2007-8-8-r177.
... Milk is the secretion of the mammalian glands, with its major role in providing nutrients and immunological protection to infants and adults (Oftedal, 2012). It contains a myriad of ingredients around 87% water, 3% protein, 4e5% lactose, 3e4% fat, 0.8% minerals, and 0.1% vitamins (Haug, Høstmark, & Harstad, 2007). ...
... After fresh milk, milk powder (MP) is the second most important food item. It is manufactured by drying milk (Skanderby, Westergaard, Partridge, & Muir, 2009), which was developed as a method of preserving milk during World War II as a way of preventing microorganism development, ensuring a constant supply in the event of a shortage, and reducing weight, which made transportation and storage easier (Oftedal, 2012). From 2013 to 2016, Fonterra Co-operative Group Limited built the world's largest spray dryers for MP, capable of generating 30 tonnes of MP per hour, indicating a trend to increase production (Burke et al., 2018). ...
... Secondly, the powder particles fall further on a vibrating fluidised bed where they are subjected to a stream of warm air at 80e120 C to leave the milk semi-dried particles with 2e4% moisture content. Finally, the dried milk particles fall onto a static fluidised bed before being cooled to avoid particle agglomeration (Oftedal, 2012). ...
Milk powders (MPs) have become one of the most significant dairy products consumed globally; consequently, ensuring product quality and safety is critical. It is expected that better milk adulteration detection techniques will continue to be developed, implemented, and disseminated. In this review, we present a comprehensive snapshot production of MPs, ways of enhancing their quality, and the analytical methods used to ensure safety from adulterants and contaminants. Spray drying is a powerful tool for improving the shelf life of milk by converting it into MPs. Despite these advantages, technological processes affect the physicochemical properties of MPs in comparison with fresh milk. The many aspects such as adulterants, contaminants, authentications, etc., that impact the quality and safety of products are dissected. Finally, different analytical methods for the rapid detection of dried milk adulterants and contaminants are reported, highlighting their advantages and limitations in ensuring dried milk safety and high quality.
... Additionally, care-associated materials can be deposited outside porous soft-shelled eggs [42], secreted or excreted by glands in synchronous or asynchronous transfers [5,31], offspring can cannibalize their siblings or even the parents [51,55,56], or the parent can lay trophic eggs for offspring to eat [57]. Mucus is used for feeding offspring in many aquatic taxa [58][59][60], and many animals have even evolved specialized feeding organs such as mammary glands or larval tubercles [61,62]. All forms of viviparity likely include social transfers, from invertebrates like tsetse flies to eutherian mammals with their highly specialized placentas [63,64]. ...
... The amniotic egg was an evolutionary innovation that allowed for greater maternal provisioning through the egg. Synapsids evolved glandular skin, a critical pre-adaptation that would go on to be necessary for the evolution of milk[4,61]. Therapsids laid their eggs in a burrow and provided parental care[129], another important pre-adaptation. ...
Full-text available
Since the dawn of life, transfers of metabolized material between individuals have led to great innovations of evolution. When metabolized material is transferred from one individual’s body to another (as with sperm, eggs, milk, symbionts), secondary manipulative molecules that induce a physiological response in the receiver are often transferred along with the primary cargo. The bioactive and transfer-supporting components in these socially transferred materials have evolved convergently to the point where they can be used in applications across taxa and type of transfer. Because these materials’ composition is typically highly dynamic and context-dependent, their focused study will allow deeper understanding of their transformative evolutionary and physiological role. We synthesize a conceptual framework for their study, and discuss future directions.
... In the context of romantic love and pair bonds, the attachment system did not evolve to facilitate bi-540 parental care as bi-parental care is a later consequence of a socially monogamous repertoire in 541 primates (Opie et al., 2013) and mammals (Lukas & Clutton-Brock, 2013), more generally. The 542 attachment system, as it relates to maternal care in our lineage, may have evolved hundreds of 543 millions of years earlier when mamillary glands started to produce milk-type substances and mother-544 infant bonding was needed to facilitate care of infants by pre-mammalian synapsid mothers (see 545 Oftedal, 2012). It has been co-opted numerous times to form the variety of close social relationships 546 associated with mammals (Broad et al., 2006). ...
For almost 25 years, the predominate evolutionary theory of romantic love has been Fisher’s theory of independent emotion systems. That theory suggests that sex drive, romantic attraction (romantic love), and attachment are associated with distinct neurobiological and endocrinological systems which evolved independently of each other. Psychological and neurobiological evidence, however, suggest that a complementary theory requires attention. A theory of co-opting mother-infant bonding sometime in the recent evolutionary history of humans may partially account for the evolution of romantic love. I present a case for this theory and a new approach to the science of romantic love drawing on human psychological, neurobiological, and (neuro)endocrinological studies as well as some animal studies. The hope is that this theoretical review, along with other publications, will generate debate in the literature about the merits of the theory of co-opting mother-infant bonding and a new evolutionary approach to the science of romantic love.
... LF has a direct bactericidal effect on common respiratory tract infection bacteria, such as Actinobacillus pleuropneumoniae and Legionella pneumophila [21,22]. LF inhibits the growth and proliferation of iron-demanding bacteria by binding with iron [23]. The peptide fragment decomposed by LF has a higher antibacterial activity than natural LF, and its antibacterial mechanism is still not perfect [24]. ...
Full-text available
Background: Pulmonary tuberculosis (PTB) affects patients' lives and reduces their quality of life. Lactoferrin (LF) is a member of the transferrin family. We explore the expression level of LF in the peripheral blood of PTB patients and the clinical significance of LF in the diagnosis and course evaluation of PTB. Methods: Plasma LF concentration and neutrophil LF concentration were measured, and their correlation was analyzed, as well as their diagnostic validity and specificity for PTB. Results: Plasma LF concentration of active PTB patients was higher than that of stable PTB patients and patients in the control group. Neutrophil LF concentration of active PTB patients was higher than that of the control group, whereas it was reversed in stable PTB patients. However, there was no significant difference in the levels of neutrophil LF and plasma LF between primary and retreated PTB patients. There was a positive correlation between them, and the LF level was positively correlated with the TB replication load. Neutrophil LF had a higher specificity and sensitivity for diagnosing stable PTB, while plasma LF had higher specificity and sensitivity in the diagnosis of active PTB. Conclusions: Plasma LF has a higher sensitivity and specificity for the diagnosis of active PTB, and LF might be a biological indicator of PTB.
... [25][26][27] Non-nutritive bioactive compounds in milk enhance offspring survival by promoting the healthy development of the immune system and intestinal microbial colonization. 2,14,[28][29][30][31] Emerging evidence also suggests that these bioactive components in milk may help protect infants against acute infections, neuroinflammation and systemic inflammation, and metabolic diseases, including obesity, hypertension, and/or type I and II diabetes. 1,2,12,32,33 There is considerable evidence that perinatal (e.g., the combined period of prenatal and postnatal life) maternal overnutrition and psychosocial stress play a role in shaping metabolic and neurodevelopmental outcomes in the offspring. ...
Full-text available
Lactation is a critical time in mammalian development, where maternal factors shape offspring outcomes. In this scoping review, we discuss current literature concerning maternal factors that influence lactation biology and highlight important associations between changes in milk composition and offspring outcomes. Specifically, we explore maternal nutritional, psychosocial, and environmental exposures that influence non-nutritive bioactive components in milk and their links to offspring growth, development, metabolic, and behavioral outcomes. A comprehensive literature search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Extension for Scoping Reviews (PRISMA-ScR) guidelines. Predetermined eligibility criteria were used to analyze 3,275 papers, and the final review included 40 primary research articles. Outcomes of this review identify maternal obesity to be a leading maternal factor influencing the non-nutritive bioactive composition of milk with notable links to offspring outcomes. Offspring growth and development are the most common modes of programming associated with changes in non-nutritive milk composition due to maternal factors in early life. In addition to discussing studies investigating these key associations, we also identify knowledge gaps in the current literature and suggest opportunities and considerations for future studies.
... Human milk is nutritionally superior to other liquids and solid foods for infants [9,10]. It is a biological fluid that contains nutritive and non-nutritive components [11][12][13] which are essential for optimal growth and development of infants [14][15][16][17][18][19]. It contains bioactive compounds that help in developing the immune system and intestinal microbiota [20,21], thus providing protection against gastrointestinal, and respiratory infections [22,23] along with allergic diseases [24][25][26]. ...
Full-text available
The purpose of this systematic review was to understand Indian mothers’ milk composition and report changes in it over the past 100 years. A review was conducted in accordance with PRISMA and registered with PROSPERO (CRD42022299224). All records published between 1921 and 2021 were identified by searching databases Google Scholar, ResearchGate, PubMed, and the Cochrane Database of Systematic Reviews. All observational, interventional, or supplementation studies reporting macronutrients (protein, fat, lactose) in milk of Indian mothers, delivering term infants, were included. Publications on micronutrients, preterm, and methods were excluded. Milk was categorized into colostrum, transitional, and mature. In all, 111 records were identified, of which 34 were included in the final review. Fat ranged from 1·83 to 4·49 g/100 mL, 2·6 to 5·59 g/100 mL, and 2·77 to 4·78 g/100 mL in colostrum, transitional, and mature milk, respectively. The protein was higher in colostrum (1·54 to 8·36 g/100mL) as compared to transitional (1·08 to 2·38 g/100 mL) and mature milk (0·87 to 2·33 g/ 100 mL). Lactose was lower in colostrum (4·5–6·47 g/100 mL) as compared to transitional (4·8–7·37 g/100 mL) and mature milk ranges (6·78–7·7 g/100 mL). The older studies (1950–1980) reported higher fat and protein in colostrum as compared to subsequent time points. There were variations in maternal nutritional status, diet, socioeconomic status , and regions along with study design specific differences of time or methods of milk sampling and analysis. Additionally, advancements in methods over time make it challenging to interpret time trends. The need for conducting well-designed, multicentric studies on nutrient composition of Indian mother’s milk using standardized methods of sampling and estimation for understanding the role of various associated factors cannot be undermined.
... Some IF nutrients are related to the human immune system, such as vitamin A, Fe, and Zn. Vitamin A is essential for visual development and cell differentiation while assisting with immune system establishment (27,28). Lactoferrin can also transport Fe as an immune factor, while Zn is vital for immune cell functionality and cytokine secretion. ...
Full-text available
Since infant formula (IF) manufacturers aim to produce a product as close to breast milk as possible, fortified nutrients are usually added. Generally, an IF is produced by adjusting the types and proportions of vitamins and minerals. This study comparatively examined the content of the six nutrients in different compound forms in vivo and evaluated the effect of different nutrient pack groups on immunity and growth. The results indicated that the simulated-human milk nutrients [minerals zinc (Zn), iron (Fe), calcium (Ca), and vitamins A, E, and B 1 ] were more easily absorbed by the body while effectively regulating immunity. This study provides a scientific foundation for developing, manufacturing, and applying imitation-breast formula milk powder.
Homeostasis during lactation is a special case in which the unit for regulation is a dyad comprising the mother and her currently nursing offspring (the mother–offspring dyad). This arrangement is not a trivial. A litter of mice can have a mass greater than the mother and nutrient demands that far exceed her. Homeostasis for milk secretion, appetite, and calcium metabolism must come under integrated control, responding seamlessly to the needs of the mother and the offspring. Serotonin (5-HT) is a primary local regulator of mammary homeostasis. 5-HT synthesis in the mammary epithelium is high during lactation and increases during milk stasis. Two important functions are attributed to the 5-HT system. Firstly, when alveolar spaces are filled with milk 5-HT inhibits milk secretion and opens tight junctions. This feedback induces early phases of involution. Secondly, 5-HT induces synthesis and secretion of parathyroid hormone-related peptide (PTHrP).
Full-text available
Human milk is a highly complex liquid food tailor-made to match an infant's needs. Beyond documented positive effects of breastfeeding on infant and maternal health, there is increasing evidence that milk constituents also impact child neurodevelopment. Non-nutrient milk bioactives would contribute to the (long-term) development of child cognition and behavior, a process termed 'Lactocrine Programming'. In this review we discuss the current state of the field on human milk composition and its links with child cognitive and behavioral development. To promote state-of-the-art methodologies and designs that facilitate data pooling and meta-analytic endeavors, we present detailed recommendations and best practices for future studies. Finally, we determine important scientific gaps that need to be filled to advance the field, and discuss innovative directions for future research. Unveiling the mechanisms underlying the links between human milk and child cognition and behavior will deepen our understanding of the broad functions of this complex liquid food, as well as provide necessary information for designing future interventions.
Full-text available
Maintaining water balance is essential for organismal health, and lactating females must balance individual needs with milk production and offspring hydration. Primate milk is dilute and presumed to be the primary source for infant hydration for a considerable time period. Few studies have investigated the hydration burden that lactation may place on female primates. In this study, we investigated sources of variation in female and offspring drinking frequency among wild chimpanzees (Pan troglodytes). We hypothesized females would experience seasonal and lactation hydration burdens and adjust their drinking behavior to accommodate these, but this hydration burden would vary between females of different dominance ranks. We also predicted that parity would relate to maternal drinking frequency since primiparous females are still investing in their own growth. Finally, we predicted that offspring would drink more in the dry season and as they aged and lost milk as a water source, but that offspring of high-ranking females would be buffered from these effects. Using 41 years of long-term data on the behavior of mothers and offspring of Gombe National Park, we found that mothers drank more in the dry season, but there was no significant difference between mothers of different ranks during this period. Low-ranking females drank significantly more than mid- and high-ranking females during late lactation. Offspring also drank more in the dry season and as they aged, but there was no evidence of buffering for those with high-ranking mothers. While chimpanzees in our study population drank infrequently, they do demonstrate noticeable shifts in drinking behavior that suggests seasonal and reproductive hydration burdens.
Full-text available
The evolutionary origin of the mammary gland has been difficult to establish because little knowledge can be gained on the origin of soft tissue organs from fossil evidence. One approach to resolve the origin of lactation has compared the anatomy of existing primitive mammals to skin glands, whilst another has examined the metabolic and molecular synergy between mammary gland development and the innate immune system. We have reviewed the physiology of lactation in five mammalian species with special reference to these theories. In all species, milk fulfils dual functions of providing protection and nutrition to the young and, furthermore, within species the quality and quantity of milk are highly conserved despite maternal malnutrition or illness. There are vast differences in birth weight, milk production, feeding frequency, macronutrient concentration, growth rate and length of lactation between rabbits, quokkas (Setonix brachyurus), pigs, cattle and humans. The components that protect the neonate against infection do so without causing inflammation. Many protective components are not unique to the mammary gland and are shared with the innate immune system. In contrast, many of the macronutrients in milk are unique to the mammary gland, have evolved from components of the innate immune system, and have either retained or developed multiple functions including the provision of nourishment and protection of the hatchling/neonate. Thus, there is a strong argument to suggest that the mammary gland evolved from the inflammatory response; however, the extensive protection that has developed in milk to actively avoid triggering inflammation seems to be a contradiction.
During lactation, marine mammals such as seals (Phocidae), fur seals and sea lions (Otariidae), manatees (Sirenia), and dolphins and whales (Cetacea) produce exceptionally rich milks containing high but changing levels of fat and energy, and low levels of sugar. The milk is lower in protein, relative to energy content, than the milk of related terrestrial species. The primary evolutionary factor favoring high-fat, high-energy milk appears to be the thermoregulatory demands of a marine environment, but a need to minimize maternal gluconeogenesis in species that fast during lactation or to minimize mammary storage volume in species that undergo long foraging trips without nursing may contribute to the evolution of ultrahigh-fat (40–60%) milks. Milk lipids of marine mammals are typically high in long-chain polyunsaturated fatty acids. Milk proteins include caseins and whey proteins, although otariids do not synthesize α-lactalbumin and produce milks devoid of lactose. The loss of α-lactalbumin may relate to the ability of otariids to sustain lactation despite extreme intersuckling intervals. The milks of most phocids, cetaceans, and polar bears contain low levels of various oligosaccharides plus lactose. Phocids and otariids are unusual among mammals in producing milk containing less calcium than phosphorus (Ca:P <1.0), but other marine mammals produce milk that typically has Ca:P >1.0.
Cell secretion is an important physiological process that ensures smooth metabolic activities, tissue repair and growth and immunological functions in the body. It occurs when the intracellular secretory materials are released to the exterior; these may be in the form of lipids, protein or mucous and may travel through a duct system or via blood to reach the target organ. To date three types of secretory mechanisms have been characterized, they include apocrine, holocrine and exocytosis. Apocrine secretion occurs when the release of secretory materials is accompanied with loss of part of cytoplasm. The secretory materials may be contained in the secretory vesicles or dissolved in the cytoplasm that is lost during secretion. In holocrine secretion, the entire cell is secreted into the glandular lumen, and it is presumed that the intended secretory materials are contained in the cell cytoplasm. Exocytosis is the most commonly occurring type of secretion; here the secretory materials are contained in the secretory vesicles and released without loss of cytoplasm. Apocrine secretory mechanisms have not been properly discussed; for example the biochemical and physiological pathways that regulate apocrine secretory process are not clearly known. Similarly, the plasma membrane dynamics during apocrine secretion has not been researched. In other glands morphological features during apocrine secretion have not been documented. The current paper reviews what is known about apocrine secretion, recent findings and highlights on the unresolved areas for future research.