Bioactive Functions of Milk Proteins: a Comparative Genomics Approach

Abstract

The composition of milk includes factors required to provide appropriate nutrition for the growth of the neonate. However, it is now clear that milk has many functions and comprises bioactive molecules that play a central role in regulating developmental processes in the young while providing a protective function for both the suckled young and the mammary gland during the lactation cycle. Identifying these bioactives and their physiological function in eutherians can be difficult and requires extensive screening of milk components that may function to improve well-being and options for prevention and treatment of disease. New animal models with unique reproductive strategies are now becoming increasingly relevant to search for these factors.

Full text

Bioactive Functions of Milk Proteins: a Comparative
Genomics Approach
Julie A. Sharp
1,3
&Vengama Modepalli
2
&Ashwanth Kumar Enjapoori
2
&
Swathi Bisana
1
&Helen E. Abud
3
&Christophe Lefevre
2,4,5,6
&Kevin R. Nicholas
3
Received: 20 March 2015 /Accepted: 19 June 2015 / Published online: 27 June 2015
#Springer Science+Business Media New York 2015
Abstract The composition of milk includes factors required
to provide appropriate nutrition for the growth of the neonate.
However, it is now clear that milk has many functions and
comprises bioactive molecules that play a central role in reg-
ulating developmental processes in the young while providing
a protective function for both the suckled young and the mam-
mary gland during the lactation cycle. Identifying these bio-
actives and their physiological function in eutherians can be
difficult and requires extensive screening of milk components
that may function to improve well-being and options for pre-
vention and treatment of disease. New animal models with
unique reproductive strategies are now becoming increasingly
relevant to search for these factors.
Keywords Mammary gland .Milk .miRNA .Marsupial .
Monotreme .Bioactive
Abbreviations
WFDC2WAP Four-disulfide Core Domain 2
MLP Monotreme Lactation Protein
AMP Antimicrobial Protein
LLP Late Lactation Protein
WAP Whey Acid Protein
ELP Early Lactation Protein
JEOL JEM 2100 Transmission Electron Microscopy
NEC Necrotizing Enterocolitis
Introduction
Understanding the composition of milk has been a fundamen-
tal topic in lactation biology. The molecular basis of mammary
gland functionhas been studied from an evolutionary perspec-
tive to understand the evolution of mammals and adaption of
the lactation system to support the survival of the newborns by
providing nutrients [1,2]. Milk is known as a well-balanced
source of nutrition, providing essential carbohydrates, lipids
and proteins to support the neonate. Milk is well established
nutritional source that also comprises various bioactive com-
ponents supporting early development of the neonate [3–5]
and also regulates mammary gland development and function
[6,7]. Recent studies have recognized the biofunctional po-
tential of milk in supporting the development of the newborn
by delivering bioactives [8]. Biologically active components
within milk include growth factors, proteins, peptides, fatty
acids, hormones and miRNA [4,9]. The presence of various
sources of bioactivity in milk raises questions about their role
in supporting neonatal growth and development. Studies on
the role of milk in neonates have shown that factors in milk
have a potential role in maturation of the immune system of
the neonate [10]. Milk is a source of different growth factors
*Julie A. Sharp
Julie.Sharp@deakin.edu.au
1
Institute for Frontier Materials, Deakin University, Geelong 3216,
Australia
2
School of Medicine, Deakin University, Geelong 3216, Australia
3
Department of Anatomy and Developmental Biology, Monash
University, Clayton 3800, Australia
4
Division of Bioinformatics, Walter and Eliza Hall Medical Research
Institute, Melbourne 3000, Australia
5
Peter MacCallum Cancer Research Institute, East Melbourne 3002,
Australia
6
Faculty of Science, University of Melbourne, Parkville 3010,
Australia
J Mammary Gland Biol Neoplasia (2014) 19:289–302
DOI 10.1007/s10911-015-9331-6

like epidermal growth factor, transforming growth factor,
nerve growth factor, insulin, and insulin like growth factors
supporting development [4]. Growth factors and hormones
have been observed to have a crucial role in regulating organ
development like maturation of the intestinal epithelium for
the preparation of solid food intake following birth [11]. Tra-
ditionally, it was believed that the development of the gut was
not influenced by factors in milk, but recent observations have
shown that bioactive components in colostrum and milk can
influence the development of the gastrointestinal tract [12].
The secretion of milk is a defining feature of all mammali-
an species [13]. The evolution of unique anatomical secretory
mammary glands and development of the lactation process
has seen the evolutionary success of mammals over million
years [14,15]. The term mammalia comes from the latin word
mamma (breast or mammary gland) coined by Carl Linnaeus
in 1758 [16]. A dependency on milk is key to the life history
of all mammals and is evolutionarily ancient in origin [13].
Pond (1984) summarized the process of lactation as a means
by which lactating females can convert their nutrient reserves
for milk production and offer substantial selective advantages
to feed dependent young when food is unavailable, and is a
universal feature of mammalian reproduction. Lactation pro-
vides the essential nutrients required by the smallest and most
altricial neonate to the largest and most precocial offspring
[13](Fig.1). The ability to provide easily digestible, balanced
nutritional components supported evolution of different devel-
opmental and reproductive strategies [13]. In the ancestral
history of mammalian evolution, milk has evolved not only
for nutritional value but also the earliest mechanism through
which mothers signal biochemically to their offspring by pro-
viding factors in milk that play a significant part in improving
health of the young [17–21].
Although the lactation cycle is a common feature of all
mammals, the monotremes and marsupials have evolved a
reproductive strategy that is very different from that of most
eutherians. Eutherians have a long gestation relative to their
lactation period, and the composition of milk doesn’tchange
substantially. This contrasts with reproduction and lactation in
monotremes, such as platypus and echidna, and the marsu-
pials, such as the tammar wallaby (Fig. 2). Deciphering the
functions of milk and discovery of new functional compo-
nents involved in the development of various organs requires
smart, innovative and strategic approaches. One such ap-
proach is the use of comparative models with extreme adap-
tations to lactation such as the monotreme and marsupial
whose young are born highly underdeveloped. When com-
bined with technology platforms that include genomics, pro-
teomics and bioinformatics these powerful models can be
exploited to identify milk proteins (and potentially
microRNA) with bioactivity and subsequent application as
nutraceuticals and pharmaceuticals.
Fig. 1 Developmental differences in newborn offspring of mammalian
lineages. Monotreme and marsupial young begin life as altricial young,
while most eutherian species are born comparatively well developed. a
From left: echidna adult, hatchling emerging from egg and young bFrom
left: tammar wallaby adult, newborn and pouch young cFrom left: human
adult, fetus and newborn
Fig. 2 Schematic representation of differences in length of mammalian
gestation and lactation between lineages. Monotreme and marsupials
have a short gestation and young are highly altricial at birth. This is
followed by a prolonged period of lactation. In contrast, eutherians
evolved longer gestation periods to allow greater in utero development
for the birth of a more highly developed young. The following lactation
period is comparatively short
290 J Mammary Gland Biol Neoplasia (2014) 19:289–302

Evolutionary Diversity in Mammalian Lactation
Prototherian, metatherian and eutherian mammals differ prin-
cipally in their strategy of reproduction such as gestation pe-
riod, composition of milk and length of lactation period.
Prototherian mammals comprise the extant species platypus
(Ornithorhynchus anatinus) and 2 genera of echidnas, short-
beaked echidna (Tachyglossus aculeatus)andthelong-beaked
echidna (Zaglossus spp.), which are confined to Australia and
New Guinea [22]. Monotremes have a unique position in
mammalian evolution with a highly specialized reproductive
strategy which facilitates both egg-laying and lactation char-
acterized by the secretion of complex milk [23]. Monotremes
lay eggs after a short gestation period and the egg is incubated
in a burrow (platypus) or pouch (echidna), an immature young
hatches from the egg, and therefore these highly altricial
hatchlings depend entirely on milk secreted by the mother
for development and immune protection [24]. The mother
has a long lactation period extending to 114–145 days in
platypus [25] and 160–210 days in echinda [26,27], depend-
ing on geographical location. In comparison, marsupials com-
prise over 300 diverse species distributed into two groups
called Ameridelphia and Australidelphia. Marsupials survive
mainly in Australia (~200 species, including tammar wallaby,
possums, kangaroos and koalas). The Virginia opossum is the
only extant species that can be found in North America but
several other species are located in South America (including
more than 65 species of opossums and shrew opossums) [28].
All marsupials have a brief intrauterine life (for example
28 days in the tammar wallaby) supported by a short-lived,
less developed placenta and give birth to an altricial young
which relies on an elaborate and sophisticated lactation period
for developmental cues [10,29]. The tammar wallaby is one
of the most studied marsupials, and its lactation period is di-
vided into three phases (phase 2A, phase 2B and phase 3)
based on the composition of the milk, and growth and behav-
ior of the young. Tammar young are born after 28 days of
gestation and weigh approximately 440 milligrams [30]. Dur-
ing the early phase of lactation (phase 2A), the young is per-
manently attached to the teat and the mother secretes dilute
milk with a low concentration of protein and lipids but a high
concentration of carbohydrates [31]. In the tammar, the ex-
pression of phase specific proteins include early lactation pro-
tein (ELP), whey acid protein (WAP) and late lactation
protein-A &B (LLPs) (Fig. 3)[32]. During the first 100 days,
the development of the neonate is similar to a late stage eu-
therian fetus. Therefore, the signalling factors involved in the
development of the eutherian fetus may be delivered in the
milk secreted by marsupials [33].
In general, pouch young are born with immature organs
due to their short gestation period and during early lactation,
the organs within the pouch young develop according to the
necessity for their survival. This includes developing organs
such as lungs [34], lymphoid tissues [35], nervous system
including brain and spinal cord [36,37] that all develop rap-
idly during this period of milk consumption. In mid-lactation
the young remains in the pouch without being permanently
attached to the teat and milk composition remains similar al-
though changes in milk protein composition have been ob-
served [38]. At this stage, the eyes of the pouch young are
opened, fur is visible and by end of phase 2B, the kidney is
functional toproduce concentrated urine and the thyroid gland
is fully developed [39]. By the end of lactation in phase 3, the
pouch young begins feeding on vegetation but intermittently
consumes milk from the mother until 300–350 days of age. In
this phase, the milk becomes rich in protein and lipids with a
reduction in the concentration of carbohydrates [40].
The eutherian mammals are represented by almost 4700
extinct and extant genera [41]. Eutherian mammals evolved
with a deeply invasive placenta to support in utero develop-
ment after which the mother gives birth to a well-developed
offspring compared to monotremes and marsupials [42]. Eu-
therian milk remains relatively unchanged throughout lacta-
tion apart from the initial colostrum [43]. Thus, there are sig-
nificant differences in reproductive strategies among three
groups of mammals but the common feature that unites these
divergent reproduction strategies is that all mammals secrete
milk to supply nutrition and bioactive components to the new-
born until they become independent at weaning [44]. Oftedal
(2002) proposed that mammary gland secretions first originat-
ed from glandular skin secretions in synapsids which laid
parchment-shelled eggs which occurred well before the
Fig. 3 Lactation cycle of the tammar wallaby showing changes in milk
composition during different phases of lactation (phase 2A, phase 2B and
phase 3) and growth of the young. The expression of ELP (early lactation
protein),WAP (whey acidic protein) and LLP (Late lactation protein) was
observed only at specific phases. Modified from [10]
J Mammary Gland Biol Neoplasia (2014) 19:289–302 291

common ancestor of living mammals appeared on the earth
[15,23]. The early amniotes (sauropsids and synapsids) in
general laid ancestral parchment-shelled eggs; however,
sauropsids evolved to develop calcified eggshells resistant to
moisture loss when exposed to vapor pressure gradients [13].
In contrast, synapsids did not evolve calcified eggshells but
instead produced glandular skin secretions to prevent eggs
from drying under ambient conditions during the course of
incubation [15,23]. These glandular skin secretions are sug-
gested to have evolved from the primary function of providing
moisture and antimicrobials to parchment-shelled eggs to a
new function associated with the supply of nutrients and other
components like antimicrobials for the young [15]. The egg-
laying ability of synapsids was retained in early mammals
which include the monotremes that lay parchment-shelled
eggs and produce mammary gland secretions to provide nu-
trients for the young [45,46].
Identifying the Potential of Milk as a Source
of Bioactives to Support Development of the Suckled
Yo u n g
Bioactive Milk Peptides
Peptides derived from milk proteins constitute a major family
of signalling molecules [47].Recent studies have revealed that
different fractions of milk protein provide groups of specific
peptides including, casein phosphopeptides, alpha lactorphins
and beta lactorphins, antihypertensive peptides, casokinins,
glycomacropeptide and opioid peptides [48,49]. These
milk-derived peptides have nutraceutical, antimicrobial, anti-
oxidative, antiviral and antihypertensive functions [48,50,51]
and are beneficial to the function of a variety of organs includ-
ing the gastrointestinal, immune, cardiovascular and nervous
systems [52].
Milk proteins are broken down to fragments by the action of
enzymes derived from secretions within the stomach and small
intestine during digestion [47–49,53]. However, in the case of
tammar pouch young the digestive system is very underdevel-
oped at the time of birth and only matures during mid-lactation
phase2B (100–200 days) [54]. As an alternative, tammar milk
may contain enzymes that support digestion and recent studies
have demonstrated that endogenous enzymatic activities in-
cluding amylase, catalase, lactase, lactoperoxidase, lipase and
phosphatase act on milk proteins to release bioactive peptides
[55,56]. Hence, it is conceivable that tammar milk may include
specific enzymes to hydrolyse milk proteins to produce bioac-
tive peptides at specific times during the lactation cycle as an
additional mechanism to support neonatal development. Inter-
estingly, endogenous human milk peptides have been found to
be greater in milk produced by mothers of preterm infants
compared to term infants [57]. Further studies are needed to
focus on understanding tammar milk derived enzymes and the
potential bioactive peptides derived from fragmentation.
Milk MiRNAs: Potential New Regulators of Newborn
Development
Recent studies have shown milk also contains other compo-
nents such as miRNAs [9,11] indicating that in addition to
proteins and peptides, miRNA may be a new class of signal-
ling molecules that contribute to the development of the neo-
nate. Milk miRNAs may have a crucial role in development
due to their key role in gene regulation [58]. Together with
proteins and peptides, the mammary gland also packages and
secretes miRNAs [59]. MicroRNAs are small untranslated
RNAs involved in post-transcriptional modification of
mRNAs, which in turn regulates protein levels [60]. miRNAs
play a crucial role in regulating a wide range of cellular func-
tions, such as cell differentiation, proliferation and cell death
[61,62]. Recent studies have shown that secretory miRNAs
are found in body fluids including breast milk [59], saliva
[63], plasma [64] and urine [65]. The presence of miRNAs
in these body fluids suggests they can act as paracrine signals
to regulate gene expression in adjacent cells/tissues by
allowing cell to cell signalling and subsequently regulating
cellular function. Discovery of miRNA in the milk of various
species potentially has profound implications in understand-
ing their role in neonate development and immune regulation.
However, the precise mechanism of secretion and the physio-
logical role of these miRNAs is not yet clear. Studies on the
miRNA expression profiles of human breast milk have shown
the presence of functional miRNAs, indicating that milk me-
diates the supply of maternal miRNA to infants, potentially
further regulating the gene expression within infant tissues
during lactation [11]. The existence of exogenous miRNAs
in the blood serum and other body fluids may suggest that
miRNAs can travel throughout he circulation and play a sig-
nificant role in other sites of the body [66,67]. Recent studies
on transportation of milk miRNAs in different eutherian spe-
cies have shown that miRNAs are packed into the exosomes
and passed on to neonates along with other components [11,
68,69]. Comparative studies on milk miRNA from different
eutherian species includes human [11], porcine [68] and goat
[69]. However, their functional role within the neonate has not
been clearly defined but a potential immunological role in the
newborn has been suggested [59].
The miRNA knockout studies in both laboratory models,
zebrafish and mouse have shown miRNAs are involved in
many biological process, including organogenesis and mor-
phogenesis during fetal development including liver [70–72],
brain [70,73,74], kidney [75] and retina [76,77]byregulat-
ing mRNA expression. The latest reports on expression pro-
files of miRNAs in the developing lung suggest a role in
292 J Mammary Gland Biol Neoplasia (2014) 19:289–302

regulating lung development [78]; for example miR-127 has
been implicated in regulating terminal bud development [79].
Work to date on milk miRNAs has identified their presence
in milk but their functional role in neonatal development still
needs to be comprehensively analyzed. The existence of
miRNAs in milk supports the concept that milk miRNAs could
represent new signalling agents in the molecular relationship
between mother and child that has evolved in the mammal
kingdom through lactation. The monotreme and marsupial are
ideal models to investigate the role of milk miRNAs in neonatal
development. Recently we showed that tammar milk contained
a substantial amount of miRNAs involved in developmental
processes (Table 1) along with other RNA molecules [9]. These
miRNAs were specifically transported in exosomes (Fig. 4a),
as shown in milk from eutherian species [11,68,69,91].
Exosomes (Fig. 4b, c) and miRNA have also been identified
in platypus and echidna milk (Enjapoori unpublished) suggest-
ing these evolved early in the evolution of milk production.
Further analysis of tammar exosomal miRNAs showed they
were stable under harsh conditions, indicating that milk
miRNAs may be successfully transported to the pouch young
without degradation and may survive longer in the intestinal
tract [9] . Therefore, the presence of exosome-like vesicles in
milk suggests that these secretory vesicles may provide protec-
tive packaging and transport of miRNA to signals the neonate.
In addition, the immature intestinal epithelial lining of pouch
young allows nonspecific uptake of large molecules from milk
such as immunoglobulins for a major period of lactation
[92–94], a process which could facilitate transfer of exosomes
across the gut. This indicates that tammar wallaby milk
miRNAs are most likely transported into the pouch young
blood circulation. The high levels of miRNA secreted in milk
has allowed the identification of differentially expressed milk
miRNAs during the wallaby lactation cycle and demonstrates
the dynamics of milk miRNA profiles across the tammar wal-
laby lactation cycle [9]. This analysis identified putative
markers of mammary gland activity and functional candidate
miRNAs which may assist growth and timed development of
the young. This approach highlights the value of comparative
quantitative transcriptomics to improve understanding of milk
composition and, more importantly, has paved the way forward
to address the true potential of milk miRNA functionality and
the full impact of milk consumption on health and wellbeing.
Tabl e 1 Differentially expressed
tammar milk miRNAs and their
function during development
miRNA Function Reference
miR-148 Growth and development of normal tissues [80]
miR-184 Development of central nervous system and regulates the
balance between neural stem cell proliferation and differentiation
[70,73,74]
let-7b Neural stem cell differentiation and proliferation [81]
let-7 s Regulates the timing of terminal differentiation during embryogenesis [82,83]
miR-122 Liver-specific and key role in liver development [84][71,72]
miR-22 Neural system and erythroid development and maturation [85,86]
miR-375 Highly expressed in hormone secreting organs
(pancreas and pituitary gland) and regulates pancreas organogenesis
[87–89]
miR-204 Lens and retinal development [77,90]
miR-30 Kidney development by regulating Xenopus pronephros development [75]
Fig. 4 Exosomes in monotreme and marsupial milk. Exosomes (30–
100 nm) were prepared from atammar wallaby, b, platypus and cand
short-beaked echidna milk by differential centrifugations and visualized
by JEM 2100 transmission electron microscopy (JEOL) with an acceler-
ating voltage of 200 kV
J Mammary Gland Biol Neoplasia (2014) 19:289–302 293

Bioactivity of Milk for Immune Defence; Evidence
from the Monotreme and Marsupial
The best known antibacterial proteins family, the cathelicidins
are secreted in milk as well as epithelial surfaces and in neu-
trophils and have been proposed to provide a first line of
defence against infection by acting as natural antibiotics [95,
96]. Tammar mammary cathelicidins are secreted abundantly
in milk during early neonatal life (mid lactation) and weaning
(involution) and display antibacterial activity against the com-
mon Staphylococcus aureus,Pseudomonas aeruginosa,En-
terococcus faecalis and Salmonella enterica species [96].This
correlated with stages of lactation associated with higher risks
of infection in pouch young and mammary gland. Similarly, in
the tammar, WFDC2 (WAP four-disulfide core domain 2) ex-
pression has been identified in the mammary gland during
lactation [97]. Tammar WFDC2 is comprised of two 4-DSC
domains: domain III on the amino terminal end and domain II
at the carboxyl terminal end. The domain II of the protein
exhibited significant antibacterial activity against S. aureus, S.
enterica and P. aeruginosa while no activity was seen against
gut commensal E. faecalis [98]. The timing of WFDC2 expres-
sion during pregnancy, early lactation and involution correlates
with stages of the lactation cycle that have increased risk of
infection in the mammary gland. Lactoferrin and lysozyme
are other examples of milk bioactives present in milk. These
proteins are capable of degrading gram negative and positive
cell walls, respectively [17,99–101]. In newborn mammals,
immature intestinal immune function and the intestinal epithe-
lial lining may be susceptible to systemic infections. Secretion
of antimicrobials and functional nutrients into mother’smilkis
a potential strategy to reduce the young early-life infections,
and protect the integrity of the epithelial monolayer.
Recently two novel milk proteins, AMP (antimicrobial pro-
tein) and MLP (monotreme lactation protein) have been iden-
tified only in both platypus and echinda milk [102]. AMP and
MLP are abundantly expressed in milk at similar levels to
caseins [103,104]. Recombinant AMP protein was found to
have significant bacteriostatic activity against Escherichia
coli,S. enterica and S. aureus, and showed significant inhibi-
tion of growth of Staphylococcus epidermidis and
P. aeruginosa [103].However, AMP showed no inhibition
of growth of the bacterial species, E. faecalis demonstrating
that AMP exhibited strain-specific antibacterial activity. Sim-
ilarly, the study of recombinant MLP also showed significant
antibacterial activity against two gram-positive bacterial spe-
cies S. aureus and E. faecalis but had no effect on the third
gram-positive species, S. epidermidis, nor the gram-negative
E. coli 2348/69, P. aeruginosa,E. coli O157:H7 and
S. enterica [104]. Antimicrobial activity was observed but
levels of activity varied between S. aureus and E. faecalis.
Both AMP and MLP protein sequence are highly con-
served between the two monotreme species studied. However
homologues have not been identified in other mammals sug-
gesting that these proteins are specific to this lineage and
therefore highly specialized in their role in monotreme lacta-
tion. Monotreme mammary glands are unique because they
have no nipples. Milk produced by the monotreme emerges
from a cluster of openings of mammary gland ducts (areola or
milk patch) onto the female’s abdomen and the young suck up
the milk directly from the abdominal surface [105,106]. It is
therefore predicted that AMP and MLP may be necessary to
facilitate nippleless delivery of milk which is more prone to
bacterial exposure. A major rationale behind this hypothesis is
that highly altricial hatchlings are immunologically naïve lack-
ing differentiated lymphoid tissue and mature immune effector
cells [107]. It has been reported that monotreme do not possess
CD4 T cell memory immune response and lymph nodes, which
are characteristic of mammalian immunity [108]. The hatchling
without immunological tissues will have lower resistance to
infection from a wide range of pathogenic organisms in the
non-sterile environment of the burrow or pouch, and therefore
may require high concentrations of proteins like AMP and MLP
in the milk for anti-bacterial protection.
Mother-to-hatchling transfer of pathogens during milk in-
gestion as the young lick the abdominal fur on the milk patch
is very problematic and may present one of the most signifi-
cant challenges to the survival of the young. In marsupials
such as the tammar wallaby this disadvantage appears to have
been minimised by the development of a teat to which the
altricial young permanently attaches for the first 100–125 days
of lactation [38,109,110]. Marsupial pouch secretions also
contain antimicrobial proteins which provide immune protec-
tion to the immune deficient pouch young along with antimi-
crobial proteins in milk [10,96,98,111,112]. Interestingly,
some of the antibacterials are delivered in the milk only when
the young is immunocompromised or when the mammary
gland is particularly susceptible to infection. In eutherians,
the developing young are protected by the sterile confines of
the mother’s uterus and young are born at a more advanced
stage of development. During intra-uterine development the
immune system of the fetus becomes more developed (al-
though not to full maturity) and a level of protection is pro-
vided postpartum via colostrum and milk bioactives [113].
Therefore the evolution of a sophisticated delivery system of
signalling molecules necessary for continued development of
the young whilst maintaining the necessary protection against
threats such as bacterial infection appear to have evolved dif-
ferently in each of the mammalian lineages.
Eutherian Placenta vs Marsupial Milk: Delivery
of Bioactives to Support Development of the Young
Factors responsible for development of the eutherian fetus are
generally supplied through the placenta via the umbilical cord.
In eutherians, the placenta mediates intrauterine fetal
294 J Mammary Gland Biol Neoplasia (2014) 19:289–302

development and also plays a key role in fetal growth by
supplying various bioactives [114,115]. During fetal devel-
opment the placenta performs a wide range of physiological
functions which are subsequently performed by the neonate
after birth including respiration, endocrine and digestive pro-
cesses [116,117]. Blood from the fetus enters the umbilical
arteries of the umbilical cord and blood enters the placenta
with the required amount of oxygen and nutrition [118]. Re-
cent studies have demonstrated that the placenta plays an in-
tegral role in programing both brain and lung development of
the fetus [119–122].
In contrast the majority of marsupial young development
occurs postnatally, the associated changes are comparable to
the developmental changes in the fetus in eutherians [123].
Hence, bioactives involved in regulating eutherian fetal devel-
opment delivered to the fetus via the placenta are proposed to
be delivered through milk by the marsupial mammary gland
[54,110,124]. Early attempts to understand the role of milk in
development of tammar pouch young have shown that factors
in milk have a potential role in maturation of the immune
system of the neonate [10]. Recent studies on understanding
the changes in tammar milk protein secreted during lactation
have revealed several factors that may involve maturation of
internal organs [54,110,124].Theseincludethegrowth
hormone-like and growth factors like epidermal growth factor,
transforming growth factor, nerve growth factor, insulin, and
insulin-like growth factors which support development
[125–127]. Fostering experiments performed in tammars to
understand regulatory effects of milk composition on the rate
of pouch young development have demonstrated that transfer-
ring early pouch young to late lactating tammar mothers can
accelerate the growth and development of pouch young [54,
128]. This is presumably due to disruption of the timing of
delivery of essential bioactives necessary for growth and
development.
Milk Bioactives in Gut Development
Studies attempting to identify the role of milk factors in gut
development of tammar pouch young have shown that every
phase of milk has specific factors triggering phenotypic
changes in the neonate stomach, where the late phase milk
can drive stomach maturation following cross fostering of
the early phase neonate to late phase lactating mother [54].
Milk is a vital immune modulator aiding in regulation of the
microbial flora of neonate’s gut [129]. As previously
discussed, studies of gene expression profiles of tammar wal-
laby milk protein genes have shown differential expression
profiles of genes encoding antibacterial proteins WFDC2
[97] and cathelicidin [130]. Further analysis of the WFDC2
protein has shown specific antibacterial activity protecting the
mammary gland of the mother, whilst avoiding the disruption
of neonatal gut microbial flora [98].
Growth factors and hormones have a crucial role in regu-
lating organ development like maturation of neonate gut for
preparation of food intake [11]. Human preterm infants (30–
37 weeks) can reduce the risk of necrotizing enterocolitis
(NEC), a result of a poorly developed gastrointestinal tract,
by breast feeding [131,132]. Studies attempting to identify the
role of milk factors in gut development in the marsupial
tammar wallaby pouch young have shown that every phase
of milk has specific factors that trigger phenotypic changes in
the neonate stomach [54]. The physiological delay in gut mat-
uration is considered advantageous in mammals like marsu-
pials, as their gastro-intestinal system is not mature until the
late phase [133]. This allows the pouch young to have the
capacity to absorb macromolecules such as maternal immu-
noglobulins in milk from the gut and this capacity for en-
hanced absorption persists through a majority of pouch life
[133]. This significantly contrasts to gut closure observed in
eutherians at a much earlier stage of development [92,93,
133]. The milk proteins and peptides transmitted from the
gut lumen into the peripheral circulation may play a regulatory
role in pouch young maturation in other organs.
Milk Bioactives in Lung Development
The lungs are a primary organ that undergoes considerable
morphological change to perform adequate levels of respira-
tion in the newborn. At birth, the respiratory system of the
neonate must be mature enough to function with efficient
gas exchange. Respiratory complications are frequently seen
in premature infants and preterm death rates are high due to
incomplete development of the respiratory system. Identifying
the components responsible for lung development and accel-
erated maturation of the lung may provide new intervention
therapies to reduce preterm deaths. During early development
of the respiratory system in eutherians, several factors in-
volved in regulating specific stages of lung development are
signalled by factors secreted by the placenta which drive lung
development for postnatal function. In contrast, marsupial
young are born with immature lungs and the majority of lung
development occurs postnatally and is dependent on factors
provided in milk.
The process of lung development in all mammals is similar
[134], but differs in the perinatal and postnatal development
period. In eutherians, the majority of lung morphogenesis oc-
curs during gestation, where the placenta performs gaseous
exchange between the fetus and the mother [135], and during
this period the lung develops completely to perform gaseous
exchange after birth. In contrast, studies of lung development
in several marsupial species including bandicoot (Isoodon
macrounus)[136], Julia Creek dunnart (Sminthopsis douglasi)
[137], tammar wallaby (Macropus eugenii) and gray short
tailed opossum (Monodelphis domestica)[138]havedemon-
strated that the lungs of marsupial newborns are still under the
J Mammary Gland Biol Neoplasia (2014) 19:289–302 295

process of development and major developmental changes in
the respiratory system occur during their early postnatal life
[34].
Newborn marsupials are similar in development to an early
stage eutherian fetus and the immature lung is required to
develop rapidly to become fully functional. Unlike eutherians,
marsupials have the ability to allow the transmission of mac-
romolecules across the gut wall due to their immature gut
[133] and therefore the milk proteins and peptides transmitted
from the gut lumen into the peripheral circulation may play a
regulatory role in lung maturation. For example, in marsupial
species like the tammar wallaby and brushtail possum, the
thyroid gland is non- functional until mid-lactation [139,
140], but analysis of thyroid hormone levels in the neonate
have shown the presence of T4 & T3 thyroid hormones [39]
and the presence of thyroid hormone in the milk throughout
lactation [109]. These findings suggest that the thyroid hor-
mones in the neonate are acquired from the mother’s milk
[39]. This theory was later supported by observing the trans-
mission of radiolabelled thyroxin from mother to neonate in
brushtail possums (Trichosurus vulpecular) and bandicoots
(Isoodon macrourus) during early lactation [141].
Recently we performed the most detailed investigation of
gray short tailed opossum and tammar wallaby lung devel-
opment to date, to understand the morphological changes
during the course of early postnatal life [142]. These studies
showed lungs in the newborns were immature, transiting
from the late canalicular to saccular stage of development
(Fig. 5). As the marsupial neonate developed the lung
underwent morphological changes by forming sacs and
gradually increased the respiratory surface area, simulta-
neously the volume of lung increased and by end of lacta-
tion the lungs were well developed and similar to that ob-
served in a eutherian newborn [135,137,138]. During early
postnatal life the lungs comprised a handful of sacs and
connected with primitive air ducts and were too immature
to provide the required amount of oxygen for the young
[143–145]. However, studies undertaken to understand the
respiratory mechanism of newborn tammar and dunnarts
[146] have demonstrated respiration occurs through the skin
for a period of early postnatal life to meet oxygen demand.
This unique mechanism diminishes gradually during early
development as the neonate lungs mature to perform effi-
cient respiration [137,147]. It appears that this mechanism
was exclusively developed by these primitive mammals to
survive during their early postnatal life and this mechanism
has been lost during evolution of sophisticated respiration in
mammals. This is also consistent with respiration through
theskininprimitiveanimalslikeamphibians[148].
Based on our observation the majority of tammar pouch
young lung development is carried out during early lactation
phase 2A. In order to test the role of tammar milk in lung
development a study was performed using mouse embryonic
lungs (E-12) cultured in media with tammar skim milk col-
lected at key time points of lactation [142]. Remarkably the
embryonic lungs showed increased branching morphogenesis
when incubated with milk collected at specific time points
between approximately day 40 to 100 lactation (phase 2A)
and reduced lung development when incubated in milk from
day of 20 lactation (Fig. 6). This suggests that day 20 milk
either lacks the necessary factors to stimulate lung develop-
ment or includes inhibitors of this process at a time when
respiration occurs through the skin and is an example of timed
developmental cues delivered through milk. This concept of
the timed presentation of milk bioactives to the young for lung
development is consistent with the timed appearance of milk
bioactives that regulate gut development in the young and
protection of the young from infection [38,54,110,124]. A
cross fostering experiment was performed whereby 25 day old
wallaby pouch young were fostered onto a series of mothers at
day 15–25 of lactation (Fig. 7) until the pouch young was
45 days old (Modepalli unpublished). Extraordinarily, the fos-
tered young showed marked reduction in lung volume and
lung development compared to the control group. Notably
total body weight and head length was also reduced in fostered
young when compared with controls.
Overall, these studies have demonstrated the importance of
milk in marsupial postnatal development and showed that
milk has evolved to support the development of immature
pouch young, in addition to providing a source of nutrition.
Fig. 5 Lung development in the tammar wallaby. The majority of lung
development in the tammar wallaby occurs postnatally and progresses
through late canalicular to alveolar stages
296 J Mammary Gland Biol Neoplasia (2014) 19:289–302

Importance of These Models for Future Study to Improve
the Health Outcomes of Preterm Neonates
Preterm neonates are highly vulnerable to death due to
interruption of their normal fetal development. Human
neonates born before 37 weeks of gestation are considered
as preterm births [149,150] and are afflicted with various
health issues including respiratory complication, limited
central nervous system and also have decreased risk of
survival [151–154]. Even though the initial survival out-
comes are positive, the preterm baby may develop long
term issues that include lifelong mental retardation, poor
health, chronic diseases like hypertension, cardiovascular
and diabetes [155].
In all newborn eutherian mammals, the ability to survive in
the external environment depends on the degree of maturity at
birth. The rate of maturation and the period of gestation are
inversely proportional. A better understanding of the process
of fetal development and the molecular mechanism of organ
maturation may provide new therapeutic approaches for re-
ducing the number of preterm deaths. During fetal develop-
ment in utero, factors involved in regulating specific morpho-
logical changes are signaled by placental secretions and am-
niotic fluid in readiness for postnatal function. In contrast,
Fig. 6 Tammar milk accelerates
lung development. aMouse
E12.5 embryonic lung cultures
with tammar milk protein. bAfter
3 days (T72) lung development is
accelerated when cultured in day
60 milk and abrogated when
cultured in day 20 milk compared
to other time points. cNumber of
terminal endbuds of differentiated
lungs grown in day 20, day 60,
and day 120 milk
Fig. 7 Foster experiment. Pouch
young of the foster group only
receives milk from day 1–25 of
lactation, while the control group
receives milk from days 1–45 of
lactation
J Mammary Gland Biol Neoplasia (2014) 19:289–302 297

marsupial pouch young are born with immature organs due to
their short gestation and during early lactation the pouch
young organs necessary for survival, such as respiratory sys-
tem [34], lymphoid tissues [35], nervous system including
brain and spinal-cord [36,37] are rapidly developed and are
entirely dependent on factors provided through milk [33,38].
Therefore, the marsupial and monotreme provide exceptional
models to identify and understand the role of these milk fac-
tors in programming and stimulating fetal development.
Future studies are needed to investigate the gene expres-
sion profile in the tammar mammary gland during the lacta-
tion cycle and proteomics to identify those factors in milk
regulating lung development. Identification of bioactives
which progress or halt the development of lung may provide
therapeutic modalities, or targets for therapeutic modalities,
for the treatment of preterm babies at risk of respiratory fail-
ure. In addition, future studies understanding the importance
of tammar milk in driving the maturation of other organs in-
cluding gut, nervous system and kidney may provide signifi-
cant evidence to support preterm neonatal maturation. The
potential approaches to produce intervention therapy will not
only assist in saving lives but may also shorten the duration of
preterm infant hospitalization leading to substantially reduced
costs for hospital-based health care.
Summary
Advances in next-generation sequencing transcriptomics and
mass spectrometry proteome studies have enabled precise de-
termination of the timing of mammary gland gene expression
and protein production during the lactation cycle from diverse
mammalian lineages. RNA profiling of both mammary gland
tissue and mammary epithelial cells secreted in milk have
been used to catalogue the complete lactome and mass spec-
trometry has been used to explore mammary secreted
proteomes. The depth of proteogenomic analysis of milk from
platypus, echidna, tammar wallaby, mouse, fur seal and hu-
man lactation have enabled the discovery of a number of novel
milk protein-coding genes and proteins which are tailored to
the particular needs of each mammalian lineage. The contin-
uous change in the composition of milk in marsupials and
monotremes presents an excellent opportunity to identify
and investigate the role of milk derived factors in neonatal
maturation. Further studies in understanding monotreme and
marsupial milk may reveal those necessary signalling factors
delivered through milk which are involved in regulating de-
velopment of various organs of the young during lactation,
such as lung and gut. These unique models may offer new
opportunities for the identification of signalling molecules that
are presented to the young at a time that correlates with pre-
natal presentation of signalling factors by the placenta and
amniotic fluid for the development of a range of tissues during
eutherian foetal development.
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