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Significantly preterm and low-birthweight (LBW) babies have diminished lung and gut development, generally fail to thrive, have increased mortality and higher frequency of mature-onset disease. Mothers often cannot breastfeed, and babies receive either formula or pasteurized donor milk, which may further limit the baby's recovery. New approaches are required to manage the early stages of neonatal development. The tammar wallaby, an Australian marsupial, has a short gestation and a simple placenta, and gives birth to an altricial young equivalent to a final trimester human embryo. The neonate remains in the pouch and attached to the teat for 100 days postpartum. The mother slows growth of the young and progressively changes the composition of the milk to deliver signals for organ development, including the lung and gut. This closely resembles the relationship between the human fetus and delivery of placental and uterine bioactives. Datasets comprised of differentially expressed genes coding for secreted proteins in early lactation in the tammar mammary gland have been compared to databases produced from human placenta, amniotic fluid, colostrum and milk to identify human homologues for the putative signaling molecules for organ development. These data will be used to develop milk fortifiers for treatment of preterm and LBW babies in both the developed and the developing world., (© 2019 Nestlé Nutrition Institute, Switzerland/S. Karger AG, Basel.)

It is now clear that milk has multiple functions; it provides the most appropriate nutrition for growth of the newborn, it delivers a range of bioactives with the potential to stimulate development of the young, it has the capacity to remodel the mammary gland (stimulate growth or signal cell death) and finally milk can provide protection from infection and inflammation when the mammary gland is susceptible to these challenges. There is increasing evidence to support studies using an Australian marsupial, the tammar wallaby (Macropus eugenii), as an interesting and unique model to study milk bioactives. Reproduction in the tammar wallaby is characterized by a short gestation, birth of immature young and a long lactation. All the major milk constituents change substantially and progressively during lactation and these changes have been shown to regulate growth and development of the tammar pouch young and to have roles in mammary gland biology. This review will focus on recent reports examining the control of lactation in the tammar wallaby and the timed delivery of milk bioactivity., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Our research is exploiting the marsupial as a model to understand the signals required for lung development. Marsupials have a unique reproductive strategy, the mother gives birth to altricial neonate with an immature lung and the changes in milk composition during lactation in marsupials appears to provide bioactives that can regulate diverse aspects of lung development, including branching morphogenesis, cell proliferation and cell differentiation. These effects are seen with milk collected between 25 and 100days postpartum. To better understand the temporal effects of milk composition on postnatal lung development we used a cross-fostering technique to restrict the tammar pouch young to milk composition not extending beyond day 25 for 45days of its early postnatal life. These particular time points were selected as our previous study showed that milk protein collected prior to ~day 25 had no developmental effect on mouse embryonic lungs in culture. The comparative analysis of the foster group and control young at day 45 postpartum demonstrated that foster pouch young had significantly reduced lung size. The lungs in fostered young were comprised of large intermediate tissue, had a reduced size of airway lumen and a higher percentage of parenchymal tissue. In addition, expression of marker genes for lung development (BMP4, WNT11, AQP-4, HOPX and SPB) were significantly reduced in lungs from fostered young. Further, to identify the potential bioactive expressed by mammary gland that may have developmental effect on pouch young lungs, we performed proteomics analysis on tammar milk through mass-spectrometry and listed the potential bioactives (PDGF, IGFBP5, IGFBPL1 and EGFL6) secreted in milk that may be involved in regulating pouch young lung development. The data suggest that postnatal lung development in the tammar young is most likely regulated by maternal signalling factors supplied through milk., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: Marsupials such as the tammar wallaby (M.Eugenii) have a short gestation (29.3 days) and at birth the altricial young resembles a fetus, and the major development occurs postnatally while the young remains in the mother's pouch. The essential functional factors for the maturation of the neonate are provided by the milk which changes in composition progressively throughout lactation (300 days). Morphologically the lungs of tammar pouch young are immature at birth and the majority of their development occurs during the first 100 days of lactation., Results: In this study mouse embryonic lungs (E-12) were cultured in media with tammar skim milk collected at key time points of lactation to identify factors involved in regulating postnatal lung maturation. Remarkably the embryonic lungs showed increased branching morphogenesis and this effect was restricted to milk collected at specific time points between approximately day 40 to 100 lactation. Further analysis to assess lung development showed a significant increase in the expression of marker genes Sp-C, Sp-B, Wnt-7b, BMP4 and Id2 in lung cultures incubated with milk collected at day 60. Similarly, day 60 milk specifically stimulated proliferation and elongation of lung mesenchymal cells that invaded matrigel. In addition, this milk stimulated proliferation of lung epithelium cells on matrigel, and the cells formed 3-dimensional acini with an extended lumen., Conclusions: This study has clearly demonstrated that tammar wallaby milk collected at specific times in early lactation contains bioactives that may have a significant role in lung maturation of pouch young.

Background: Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby., Results: Results showed that high levels of miRNA secreted in milk and allowed the identification of differentially expressed milk miRNAs during the lactation cycle as putative markers of mammary gland activity and functional candidate signals to assist growth and timed development of the young. Comparative analysis of miRNA distribution in milk and blood serum suggests that milk miRNAs are primarily expressed from mammary gland rather than transferred from maternal circulating blood, likely through a new putative exosomal secretory pathway. In contrast, highly expressed milk miRNAs could be detected at significantly higher levels in neonate blood serum in comparison to adult blood, suggesting milk miRNAs may be absorbed through the gut of the young., Conclusion: The function of miRNA in mammary gland development and secretory activity has been proposed, but results from the current study also support a differential role of milk miRNA in regulation of development in the pouch young, revealing a new potential molecular communication between mother and young during mammalian lactation.

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Asynchronous concurrent lactation (ACL) is an extreme lactation strategy in macropod marsupials including the tammar wallaby, that may hold the key to understanding local control of mammary epithelial cell function. Marsupials have a short gestation and a long lactation consisting of three phases; P2A, P2B and P3, representing early, mid and late lactation respectively and characterised by profound changes in milk composition. A lactating tammar is able to concurrently produce phase 2A and 3 milk from adjacent glands in order to feed a young newborn and an older sibling at heel. Physiological effectors of ACL remain unknown and in this study the extracellular matrix (ECM) is investigated for its role in switching mammary phenotypes between phases of tammar wallaby lactation. Using the level of expression of the genes for the phase specific markers tELP, tWAP, and tLLP-B representing phases 2A, 2B and 3 respectively we show for the first time that tammar wallaby mammary epithelial cells (WallMECs) extracted from P2B acquire P3 phenotype when cultured on P3 ECM. Similarly P2A cells acquire P2B phenotype when cultured on P2B ECM. We further demonstrate that changes in phase phenotype correlate with phase-specific changes in ECM composition. This study shows that progressive changes in ECM composition in individual mammary glands provide a local regulatory mechanism for milk protein gene expression thereby enabling the mammary glands to lactate independently., (Copyright © 2013. Published by Elsevier B.V.)

We have previously shown that the gene for MaeuCath1, a cathelicidin secreted in wallaby milk is alternately spliced into two variants, MaeuCath1a and MaeuCath1b which are temporally regulated in order to provide antimicrobial protection to the newborn and stimulate mammary growth, respectively. The current study investigated the extracellular matrix (ECM) for its regulatory role in MaeuCath1 gene expression. Reverse transcription qPCR using RNA isolated from mammary epithelial cells (WallMEC) cultured on ECM showed that ECM regulates MaeuCath1a gene expression in a lactation phase-dependent manner. Luciferase reporter-based assays and in silico analysis of deletion fragments of the 2245bp sequence upstream of the translation start site identified ECM-dependent positive regulatory activity in the -709 to -15 region and repressor activity in the -919 to -710 region. Electrophoretic Gel Mobility Shift Assays (EMSA) using nuclear extract from ECM-treated WallMEC showed differential band shift in the -839 to -710 region., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The role of milk extends beyond simply providing nutrition to the suckled young. Milk has a comprehensive role in programming and regulating growth and development of the suckled young, and provides a number of potential autocrine factors so that the mammary gland functions appropriately during the lactation cycle. This central role of milk is best studied in animal models such as marsupials that have evolved a different lactation strategy to eutherians and allow researchers to more easily identify regulatory mechanisms that are not as readily apparent in eutherian species. For example, the tammar wallaby (Macropus eugenii) has evolved with a unique reproductive strategy of a short gestation, birth of an altricial young and a relatively long lactation during which the mother progressively changes the composition of the major, and many of the minor components of milk. Consequently, in contrast to eutherians, there is a far greater investment in development of the young during lactation and it is likely that many of the signals that regulate development of eutherian embryos in utero are delivered by the milk. This requires the co-ordinated development and function of the mammary gland since inappropriate timing of these signalling events may result in either limited or abnormal development of the young, and potentially a higher incidence of mature onset disease. Milk proteins play a significant role in these processes by providing timely presentation of signalling molecules and antibacterial protection for the young and the mammary gland at times when there is increased susceptibility to infection. This review describes studies exploiting the unique reproductive strategy of the tammar wallaby to investigate the role of several proteins secreted at specific times during the lactation cycle and that are correlated with potential roles in the young and mammary gland. Interestingly, alternative splicing of some milk protein genes has been utilised by the mammary gland to deliver domain-specific functions at specific times during lactation., (Copyright © 2012. Published by Elsevier Ltd.)

WAP four disulfide core domain 2 (WFDC2) is a four disulfide core (4-DSC) protein secreted in the milk of the tammar wallaby. It is comprised of two 4-DSC domains assigned domain III at the NH2-terminal end and domain II at the COOH-terminal end. The WFDC2 gene was expressed only during pregnancy, early lactation, towards the end of lactation and involution. The WFDC2 protein showed antibacterial activity against Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resided with domain II. There was no antibacterial activity detected against Enterococcus faecalis. The observed expression pattern of tammar WFDC2 and its antibacterial activity suggests a role to either reduce mastitis in the mammary gland caused by S. aureus or to protect the gut of the young at a time when it is not immune-competent. The latter effect could be achieved without disturbing the balance of commensal gut flora such as E. faecalis., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Whey acidic protein (WAP) belongs to a family of four disulfide core (4-DSC) proteins rich in cysteine residues and is the principal whey protein found in milk of a number of mammalian species. Eutherian WAPs have two 4-DSC domains, whereas marsupial WAPs are characterized by the presence of an additional domain at the amino terminus. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. We have characterized the genomic structure of tammar WAP (tWAP) gene, identified its chromosomal localization and investigated the potential function of tWAP. We have demonstrated that tWAP and domain III (DIII) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of DIII from tWAP abolishes this proliferative effect. However, tWAP does not induce proliferation of human embryonic kidney (HEK293) cells. DNA synthesis and expression of cyclin D1 and cyclin-dependent kinase-4 genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DIII. These data suggest that DIII is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy.

Mammary development and function are regulated by systemic endocrine factors and by autocrine mechanisms intrinsic to the mammary gland, both of which act concurrently. The composition of milk includes nutritional and developmental factors that are crucial to the development of the suckled young, but it is becoming increasingly apparent that milk also has a role in regulating mammary function. This review examines the option of exploiting the comparative biology of species with extreme adaptation to lactation to examine regulatory mechanisms that are present but not readily apparent in other laboratory and livestock species. The tammar wallaby has adopted a reproductive strategy that includes a short gestation (26 d), birth of an immature young, and a relatively long lactation (300 d). The composition of milk changes progressively during the lactation cycle, and this is controlled by the mother and not the sucking pattern of the young. Furthermore, the tammar can practice concurrent asynchronous lactation; the mother provides a concentrated milk high in protein and fat for an older animal that is out of the pouch and a dilute milk low in fat and protein but high in carbohydrates from an adjacent mammary gland for a newborn pouch young. This phenomenon suggests that the mammary gland is controlled locally. The second study species, the Cape fur seal, has a lactation characterized by a repeated cycle of long at-sea foraging trips (up to 28 d) alternating with short suckling periods of 2 to 3 d ashore. Lactation almost ceases while the seal is off shore, but the mammary gland does not progress to apoptosis and involution, most likely because of local control of the mammary gland. Our studies have exploited the comparative biology of these models to investigate how mammary function is regulated by endocrine factors, and particularly by milk. This review reports 3 major findings using these model animals. First, the mammary epithelial cell has an extraordinary intrinsic capacity for survival in our culture model, and the path to either function or death by apoptosis is actively driven. The second outcome is that the route to apoptosis is most likely regulated by specific milk factors. Finally, whey acidic protein, a major milk protein in some species, may play a role in normal mammary development, but that role in vivo may be limited to marsupials. Evolutionary pressure has led to changes in the structure of the protein with an accompanying change in function. Therefore, we propose that a loss of function of this protein in eutherians may relate to a reproductive strategy that is less dependent on lactation.