Maternal regulation of milk composition,milk production,and pouch young development during lactation in the tammar wallaby (Macropus eugenii )

Specific changes in milk composition during lactation in the tammar wallaby (Macropus eugenii) were correlated with the ages of the developing pouch young (PY). The present experiment was designed to test the hypothesis that the sucking pattern of the PY determines the course of mammary development in the tammar wallaby. To test this hypothesis, groups of 60-day-old PY were fostered repeatedly onto one group of host mothers so that a constant sucking stimulus on the mammary gland was maintained for 56 days to allow the lactational stage to progress 42 days ahead of the age of the young. Analysis of the milk in fostered and control groups showed the timing of changes in the concentration of protein and carbohydrate were essentially unaffected by altering the sucking regime. The only change in milk protein secretion was a small delay in the timing of down-regulation of the secretion of whey acidic protein and early lactation protein in the host tammars. In addition, the rates of growth and development of the foster PY were significantly increased relative to those of the control PY because of ingesting more milk with a higher energy content and different composition than normal for their age. The present study demonstrates that the lactating tammar wallaby regulates both milk composition and the rate of milk production and that these determine the rates of PY growth and development, irrespective of the age of the PY.     

The extracellular matrix locally regulates asynchronous concurrent lactation in tammar wallaby (Macropus eugenii)

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.     

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

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.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1012) contains supplementary material, which is available to authorized users.     

The tammar wallaby: a model system to examine domain-specific delivery of milk protein bioactives

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.     

Enzymes of galactose metabolism in livers of suckling and adult tammar wallabies (Macropus eugenii) and other marsupials

The activities of galactokinase, hexose-1-phosphate uridylyl transferase and UDPglucose 4-epimerase in homogenates of livers of two adult and 20 suckling tammar wallabies aged from 6 to 50 weeks were investigated. The activities of all three enzymes were high until 24-30 weeks post partum, after which they declined to low levels. The activities of the three liver enzymes were high in pouch young of six other species of marsupial. Comparison of the activities of the three liver enzymes in suckling tammar wallabies with those in suckling rats showed no difference between the two species in regard to galactokinase and uridylyl transferase, but the UDPglucose 4-epimerase activity in tammar wallabies was approximately double than found in rats. This may be related to the high galactose content of tammar wallaby milk compared with rat milk. In suckling tammar wallabies, the liver had higher enzyme activities than other tissues studied. It is concluded that, contrary to the suggestion of Stephens et al. (1975), pouch young marsupials are not deficient in their ability to metabolize galactose.     

Acute involution in the tammar wallaby: identification of genes and putative novel milk proteins implicated in mammary gland function

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution.     

Changes in milk protein composition during acute involution at different phases of tammar wallaby (Macropus eugenii) lactation

This study exploited the unusual lactation cycle of the tammar wallaby (Macropus eugenii) to characterise milk composition during acute involution, a time when the mammary gland is subjected to increased risk of infection. In early-lactation, tammar milk contains elevated levels of complex oligosaccharides and low protein and lipid content. Later in lactation, protein and lipid concentrations increase significantly, whereas carbohydrate content is reduced dramatically and changes to monosaccharides. Following initiation of involution at early-lactation, the carbohydrate concentration greatly decreased, while lipid and protein concentrations were elevated, suggesting that complex oligosaccharides are the major osmole in milk at this time. In contrast, involution at late lactation, when carbohydrate concentration was very low, led to an increase in the lipid concentration, but the concentration of protein was not significantly altered. This indicates that protein synthesis during acute involution at late lactation in the tammar may be down-regulated much more rapidly than during early-lactation. Analysis of milk at day 3 after the onset of involution at early-lactation identified a number of potential antimicrobials secreted at high concentrations, including lysozyme, dermcidin, polymeric immunoglobulin receptor and fragments of beta-lactoglobulin. These proteins may protect the mammary gland by minimising the risk of potential infection during involution.     

Characterization of N- and O-linked glycosylation changes in milk of the tammar wallaby (Macropus eugenii) over lactation

As one of several biologically active compounds in milk, glycoproteins have been indicated to be involved in the protection of newborns from bacterial infection. As much of the physical and immune development of the tammar wallaby (Macropus eugenii) young occurs during the early phases of lactation and not in utero, the tammar is a model species for the characterization of potential developmental support agents provided by maternal milk. In the present study, the N- and O-linked glycans from tammar wallaby milk glycoproteins from six individuals at different lactation time points were subjected to glycomics analyses using porous graphitized carbon liquid chromatography electrospray ionization mass spectrometry. Structural characterization identified a diverse range of glycan structures on wallaby milk glycoproteins including sialylated, sulphated, core fucosylated and O-fucosylated structures. 30 % of N-linked structures contained a core (α1-6) fucose. Several of these structures may play roles in development, and exhibit statistically significant temporal changes over the lactation period. The N-glycome was found to contain structures with NeuGc residues, while in contrast the O-glycome did not. O-fucosylated structures were identified in the early stages of lactation indicating a potential role in the early stages of development of the pouch young. Overall the results suggest that wallaby milk contains structures known to have developmental and immunological significance in human milk and reproduction in other animals, highlighting the importance of glycoproteins in milk.     

The expression of beta-1,3 galactosyltransferase and beta-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation

The regulation of beta-1,3 galactosyltransferase (3betaGalT) and beta-1,4 galactosyltransferase enzymatic (4betaGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two beta-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4betaGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3betaGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3betaGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17beta-oestradiol (E). The activity of 4betaGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3betaGalT and 4betaGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.     

Cross-fostering in Macropus eugenii leads to increased weight but not accelerated gastrointestinal maturation

Stomach and small intestine development was characterized in tammar wallaby (Macropus eugenii) pouch young (PY) using both morphological and immunohistological criteria. At birth, the stomach is undeveloped in comparison to the well-developed intestinal mucosa. The stomach maintains a uniform morphology in both the forestomach and hindstomach regions until the specialization of cardiac and gastric glands are seen at PY170. Parietal cells, found throughout the mucosa are downregulated in the forestomach as cardiac glandular stomach is developing prior to the transition of the offspring to a diet that includes herbage. In the small intestine, mature-type villi are present at birth but the muscularis externa is immature and undergoes significant development around PY120 onwards. We investigated the effects of changes in maternal milk on gut development in the tammar wallaby using a cross fostering approach that provided younger pouch young with older stage milk. Younger PY (average age 67 days postpartum, n = 5) were transferred onto teats vacated by older stage PY (average age 100 days postpartum, n = 6) for 34 days before gut development was assessed. In addition milk analysis was performed before and after fostering events. Cross-fostered PY animals receiving older stage milk were found to be 31% heavier than controls. There was no difference between carbohydrate and protein concentrations however, fostered PY milk had a higher concentration of lipid than that of controls that may have contributed to heavier fostered PY. No difference was found in stomach or small intestine development between these groups using the criteria employed in this study.     

The expression of β-1,3 galactosyltransferase and β-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation

The regulation of β-1,3 galactosyltransferase (3βGalT) and β-1,4 galactosyltransferase enzymatic (4βGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two β-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4βGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3βGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3βGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17β-oestradiol (E). The activity of 4βGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3βGalT and 4βGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.     

Plasma prolactin concentrations throughout lactation in the eastern quoll, Dasyurus viverrinus (Marsupialia:Dasyuridae)

Blood samples were collected throughout lactation from eight D. viverrinus, and the concentrations of prolactin in the plasma determined by an heterologous, double-antibody radioimmunoassay. For the first 45-50 days of lactation concentrations fluctuated between 2 and 80 ng/ml but were generally less than 60 ng/ml. They then increased sharply to over 120 ng/ml and greater than 200 ng/ml in some animals. Levels began to decline after 120-130 days of lactation and were less than 40 ng/ml in all animals by the time lactation had ceased, between 160 and 185 days, when the young became independent. The timing of these changes correlates well with a rapid increase in the growth rate of the young, the time at which they first relinquish the teat and remain in the nest, their first intake of solid food shortly after eruption of the first molars and specific changes in milk composition. The pattern of prolactin concentrations throughout lactation in the quoll is therefore very similar to that previously described for the tammar, Macropus eugenii, and the possum, Trichosurus vulpecula.     

Qualitative and quantitative changes in milk fat during lactation in the tammar wallaby (Macropus eugenii)

There are major quantitative and qualitative changes in the milk lipids during lactation in the tammar wallaby, Macropus eugenii. The crude lipid content of the milk is relatively low during the first 10 weeks of lactation; between 10 and 26 weeks post partum the lipid content increases gradually but after that it increases rapidly. The triglyceride fraction of the lipid at early stages of lactation contains a large amount of palmitic acid and relatively little oleic acid whereas mature milk exhibits little palmitic and much oleic acid. In the early stages of lactation fat represents 15% of the total solids and carbohydrate 55%; around 26-30 weeks post partum the carbohydrate moiety falls sharply to a level less than 2% of the solids while lipids increase to c. 60% of the solids. These changes coincide with increases in milk solids, emergence of the young from the pouch, ingestion of herbage, and fermentation of cellulose in the stomach.     

Changes in prolactin in peripheral plasma during lactation in the brushtail possum Trichosurus vulpecula

A heterologous double-antibody radioimmunoassay has been validated for prolactin in plasma and pituitary preparations of T. vulpecula. Serial dilutions of crude pituitary homogenates and plasmas from several marsupials and purified prolactin from the tammar, Macropus eugenii, showed parallel dose response curves. In both male and female possums plasma prolactin concentrations increased in response to a single intravenous injection of thyrotrophin releasing hormone. Plasma prolactin concentrations were measured in six lactating females (June-November) and in four non-lactating females (July-October). In the following year prolactin levels were also measured in 11 possums with young less than 50 days old and in 24 possums with young aged between 100 and 145 days. In early lactation prolactin concentrations were low (less than 8 ng/ml) but increased to high levels (greater than 30 ng/ml) by 120 days and remained high until about 160 days of lactation. Thereafter concentrations declined although the young continued to take milk from the mother for a further 30-50 days. The changes in plasma prolactin concentrations throughout lactation are very similar to those described for the tammar, and this unusual pattern appears to be common to marsupials. Non-lactating possums showed no consistent changes in plasma prolactin concentrations between July and October.     

Milk carbohydrates of marsupials. II. Quantitative and qualitative changes in milk carbohydrates during lactation in the tammar wallaby (Macropus eugenii)

Milk was collected at various stages of lactation from a group of tammar wallabies, M. eugenii, in which parturition had been synchronized. The milk carbohydrate was determined by a phenol-sulfuric acid method which had been modified to give equal colour yields for galactose and glucose. The mean carbohydrate content increased gradually during the first 6 months of lactation to a peak of 13 g hexose/100 ml of milk, but then fell rapidly to much lower values, over the following 2 months. Throughouth lactation, galactose was the predominant monosaccharide constituent of acid hydrolysates of the milk carbohydrate. Glucose, glucosamine, galactosamine and sialic acid were the only other monosaccharides present. Qualitative changes were investigated by gel filtration and thin-layer chromatography. During the first 6 months post partum the milk carbohydrate was composed of a variety of oligosaccharides including lactose, but from 8 months onwards it consisted mainly of free monosaccharides. Between 6 and 8 months an intermediate pattern was observed, i.e. a mixture of lower oligosaccharides and free monosaccharides. In two animals which suckled both a new-born pouch young and a young at foot, the mammary gland supplying the new-born secreted milk which was rich in oligosaccharides, whereas that supplying the young at foot produced milk in which the carbohydrates were mainly free monosaccharides, and which had a much lower carbohydrate content.     

A novel approach identified the FOLR1 gene, a putative regulator of milk protein synthesis

This study has utilised comparative functional genomics to exploit animal models with extreme adaptation to lactation to identify candidate genes that specifically regulate protein synthesis in the cow mammary gland. Increasing milk protein production is valuable to the dairy industry. The lactation strategies of both the Cape fur seal (Artocephalus pusillus pusillus) and the tammar wallaby (Macropus eugenii) include periods of high rates of milk protein synthesis during an established lactation and therefore offer unique models to target genes that specifically regulate milk protein synthesis. Global changes in mammary gene expression in the Cape fur seal, tammar wallaby, and the cow (Bos taurus) were assessed using microarray analysis. The folate receptor α (FOLR1) showed the greatest change in gene expression in all three species [cow 12.7-fold (n = 3), fur seal 15.4-fold (n = 1), tammar 2.4-fold (n = 4)] at periods of increased milk protein production. This compliments previous reports that folate is important for milk protein synthesis and suggests FOLR1 may be a key regulatory point of folate metabolism for milk protein synthesis within mammary epithelial cells (lactocytes). These data may have important implications for the dairy industry to develop strategies to increase milk protein production in cows. This study illustrates the potential of comparative genomics to target genes of interest to the scientific community.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C(16:0), which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.     

Proteomic analysis of early lactation milk of the tammar wallaby (Macropus eugenii)

We report the successful use of 2D electrophoresis, MALDI MS/MS and chemical derivatisation protocols of guanidination and sulfonation to identify over 100 protein spots present in early marsupial milk (tammar wallaby) at 40 days lactation, where a limited translated genomic database is publicly available for cross species matching and protein identification. Of the proteins identified, 25 matched to 6 existing marsupial milk protein sequences in the NCBI database; another 6 were identified with high confidence to other mammals and have not previously been identified in marsupial milk. By using chemical derivatisation, the reliable identification of a further 81 proteins was achieved. The identified proteins could be grouped into three main functional categories — transport, nutrition and immune protection. All these proteins play a potential role in determining growth and immunological protection of the highly altricial marsupial young at 40 days after birth.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C_16:0, which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.