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.     

Cross-fostering of the tammar wallaby (Macropus eugenii) pouch young accelerates fore-stomach maturation

There are two phases of fore-stomach development during the first 200 days of pouch life in tammar wallaby. For the first 170 days, the mucosa displays an immature gastric glandular phenotype that changes to a cardia glandular phenotype, which remains for the rest of the animal’s life. During this 200-day period after birth, the pouch young (PY) is dependent on maternal milk, which progressively changes in composition. We showed previously that PY cross-fostered to host mothers at a later stage of lactation accelerated development. In this study, we investigated whether cross-fostering and exposure to late lactation stage milk affected the transition to cardia glandular phenotype. In fostered PY fore-stomach, there was increased apoptosis, but no change in cell proliferation. The parietal cell population was significantly reduced, and expression of gastric glandular phenotype marker genes (ATP4A, GKN2, GHRL and NDRG2) was down-regulated, suggesting down-regulation of gastric phenotype in fostered PY fore-stomach. The expression of cardia glandular phenotype genes (MUC4, KRT20, CSTB, ITLN2 and LPLUNC1) was not changed in fostered PY. These data suggest that fore-stomach maturation proceeds via two temporally distinct processes: down-regulation of gastric glandular phenotype and initiation of cardia glandular phenotype. In fostered PY, these two processes appear uncoupled, as gastric glandular phenotype was down-regulated but cardia glandular phenotype was not initiated. We propose that milk from later stages of lactation and/or herbage consumed by the PY may play independent roles in regulating these two processes.     

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.     

Sex determination in marsupials: evidence for a marsupial-eutherian dichotomy

In this paper, we review briefly the current state of knowledge about sexual differentiation in eutherian mammals, and then describe the situation in detail in two marsupial species: the North American opossum and the tammar wallaby. The conventional explanation for the genesis of all male somatic sexual dimorphisms in mammals is that they are a consequence of the systemic action of testicular hormones. In the absence of testes, the embryo will develop a female phenotype. We present evidence for the tammar wallaby that calls into question the universal applicability of this hormonal theory of mammalian sexual differentiation. We have shown that extensive somatic sexual dimorphisms precede by many days the first morphological evidence of testicular formation, which does not occur until around the third day of pouch life. Male foetuses, and pouch young on the day of birth, already have a well-developed gubernaculum and processus vaginalis, paired scrotal anlagen, and a complete absence of mammary anlagen, whereas female foetuses and newborn pouch young have a poorly developed gubernaculum and processus vaginalis, no scrotal anlagen, and well-developed mammary anlagen. Because it seems unlikely that the male gonad could begin hormone secretion until after the Sertoli and Leydig cells are developed, our results strongly suggest that some sexually dimorphic somatic characteristics develop autonomously, depending on their genotype rather than the hormonal environment to which they are exposed. We have been able to confirm the hormonal independence of the scrotum, pouch and mammary gland by administering testosterone propionate daily by mouth to female pouch young from the day of birth; although the Wolffian duct was hyperstimulated, there was no sign of scrotal development, or pouch or mammary inhibition. When male pouch young were treated with oestradiol benzoate in a similar fashion, there was hyperstimulation of the Müllerian duct and inhibition of testicular migration and development, but no sign of scrotal inhibition or pouch or mammary development. Our results in the tammar wallaby are consistent with the earlier studies on the opossum, whose significance was not appreciated at the time. Further evidence in support of this hormonal independence comes from earlier studies of spontaneously occurring intersexes in several species of marsupial, including the opossum and the tammar wallaby. An XXY individual had intra-abdominal testes and complete masculinization of the male reproductive tract internally, but externally there was a pouch and mammary glands and no scrotum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteins in cerebrospinal fluid and plasma of the pouch young tammar wallaby (Macropus eugenii) during development

The concentrations of total protein and albumin in cerebrospinal fluid (CSF) and plasma of tammar wallaby pouch young (Macropus eugenii) from birth until leaving the pouch have been measured. Total protein in CSF increased from birth (about 240 mg/100 ml) to 15-20 days postnatal (about 400 mg/100 ml) after which it declined. Albumin showed a proportionately greater increase from around 40 mg/100 ml to over 130 mg/100 ml, followed by decline after 75 days. Total protein and albumin in plasma increased throughout the period studied. Other proteins identified in CSF and plasma were: fetuin, alpha 2-macroglobulin, transferrin, alpha-lipoprotein, beta-lipoprotein, immunoglobin G and fibrinogen. One protein was only present in early pouch young (up to about 40 days) and was presumed to be the tammar equivalent of alpha-fetoprotein.     

A quantitative study of the morphological development and bacterial colonisation of the gut of the tammar wallaby Macropus eugenii eugenii and brushtail possum Trichosurus vulpecula during in-pouch development

We compared the rates of change of various morphological parameters of the stomach, small intestine, caecum and colon of tammar wallabies and brushtail possums with body mass during in-pouch development. These were correlated with changes in the numbers of bacterial species in the various gut segments. In the pouch-young of both species, the wet tissue masses of all gut segments increased with body mass in a positively allometric manner (i.e. with a body mass exponent > 1), suggesting that the mass of each component was disproportionately low at birth, but increased disproportionately rapidly postnatally. However, the lengths of the wallaby stomach and small intestine scaled isometrically with respect to body mass (i.e. with a body mass exponent around 0.33), which may indicate that the shape of these components changes to the adult form during early neonatal development. Conversely, the length of the caecum and colon of both wallabies and possums scaled in a positively allometric manner with respect to body mass, showing area to volume compensation. This may indicate a more general pattern of disproportionately rapid postnatal enlargement in areas that are distal to the principal sites of neonatal digestion (i.e. the stomach). The numbers of bacterial species present in the various gastrointestinal segments of both species were low in animals aged 100 days or less but there was a significant increase in microbial diversity in the caecum of brushtail possums aged over 100 days. The possum caecum also showed the greatest rate of increase in wet tissue mass relative to body mass. It is postulated that caecal development may act as a nidus for establishment of communities of commensal microflora in the developing marsupial.     

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.     

Steroid hormone content of the gonads of the tammar wallaby during sexual differentiation.

The gonads of the tammar wallaby, Macropus eugenii, are sexually indifferent at birth (Day 0) despite the fact that phenotypic sexual differentiation has already commenced as evidenced by the presence of a scrotum in males and mammary anlagen in females. The seminiferous cords of the testis first become clearly recognizable on Day 2 of pouch life, and ovarian differentiation is recognizable by Day 10. To monitor the endocrine development of the gonads during sexual differentiation of the urogenital tract, we measured the steroid hormone content in 92 pools of gonads from male and female tammar pouch young from the day of birth to 206 days of pouch life. Progesterone, estradiol, and dihydrotestosterone concentrations were low (less than 0.05 ng/mg protein) in both ovaries and testes at all stages examined, and testosterone concentrations were uniformly low in ovaries. Testosterone concentrations in testes were low on Days 0-4, averaging about 0.2 ng/mg protein; they rose by Days 5-10 to an average of 0.9 ng/mg protein, remained elevated until about Day 40, and thereafter fell to values similar to those in the ovaries. The phallus and urogenital sinus were able to convert testosterone to dihydrotestosterone from the earliest stages examined (Days 10 and 11). Thus in the tammar wallaby, as in eutherian mammals, testosterone is the androgen secreted by the developing testis, and dihydrotestosterone is formed in certain androgen target tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of corpus luteum removal during gestation on parturition in the tammar wallaby (Macorpus eugenii).

Removal of the corpus luteum of pregnancy of the tammar wallaby interfered with successful parturition if carried out before Day 17 of the 27-day pregnancy. After removal at Days 17 and 21, 40% of animals gave birth but pouch young died with 24 h; if performed at Days 23 or 25, pouch young survived. However, surgery also affected sham-operated controls if performed between Days 15 and 21. In control animals, sodium pentobarbitone followed by halothane anaesthesia was the least disruptive anaesthetic procedure. This study shows that the corpus luteum has an essential role in parturition and subsequent survival of the neonate in the pouch.     

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.     

Differential expression of WNT4 in testicular and ovarian development in a marsupial

Background  

WNT4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. Using a marsupial, the tammar wallaby, in which most gonadal differentiation occurs after birth whilst the young is in the pouch, we show by quantitative PCR during early testicular and ovarian development that WNT4 is differentially expressed ingonads.     

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.     

Mammogenesis and changing prolactin receptor concentrations in the mammary glands of the tammar wallaby (Macropus eugenii)

All 4 mammary glands of the tammar wallaby showed a steady increase in weight and prolactin receptor concentration during the luteal phase of the oestrous cycle to reach a peak at oestrus. Removal of the corpus luteum abolished this mammogenesis , while pregnancy, which in this species is a day or so shorter than the oestrous cycle, had no effect. This provides an explanation for the previous finding that pregnancy is not a necessary pre-requisite for lactation in marsupials and that nonpregnant animals will lactate very successfully, provided the suckling stimulus is applied at the correct stage of the oestrous cycle. During lactation, only the gland supplying the teat to which the pouch young was attached developed and showed any further increase in prolactin receptors; the other 3 glands remained small and inactive. These results indicate the importance of the suckling stimulus and milk withdrawal on the initiation and maintenance of lactation.     

The effects of enteral ghrelin administration on the remodeling of the small intestinal mucosa in neonatal piglets

Ghrelin is a multifunctional peptide produced predominantly in the stomach, however substantial amounts have also been found in colostrum and milk. The aim of the study was to investigate the effect of exogenous ghrelin, administered intra-gastrically, on the processes of mitosis, apoptosis, autophagy, crypt fission and changes in histometry of the small intestine mucosa in neonatal pigs, fed with a milk formula. Three groups (n=6) of piglets were used in the study. The pigs were fed either milk formula (C7) or milk formula together with ghrelin, administered via a stomach tube (7.5 μg/kg body weight (BW), (LG)) and 15 μg/kg BW (HG), every 8h for 6 days. Compared to the control group (C7), feeding milk formula supplemented with ghrelin resulted in significant changes in the small intestinal morphometry and mucosa histometry. The observed changes were dependent on the dosage of hormone and the part of intestine investigated. Administration of ghrelin via the stomach tube (HG) significantly influenced epithelial cell renewal. Moreover, we demonstrated that autophagy is involved in the small intestine mucosa remodeling and ghrelin may be an important factor for its regulation. In conclusion, we found that enteral ghrelin influences the gut mucosa remodeling in a dose-related manner in the early postnatal period. Moreover in neonates, stomach activity does not interfere with the action of ghrelin in the small intestine.     

Reproductive biology of the Red-necked wallaby (Macropus rufogriseus banksianus) and Bennett's wallaby (M. r. rufogriseus) in captivity

Bennett's wallaby ( Macropus r. rufogriseus ) of Tasmania give birth from late January to early August in marked contrast to the Red-necked wallaby ( M. r. banksianus ) of mainland south-eastern Australia which produced young in all months. Within the breeding season however, the lengths of the oestrous cycle and gestation period are similar in the two forms and did not differ by more than 0.5 days. The gestation period of about 30 days extended to almost the length of the oestrous cycle of approximately 33 days. Birth was closely followed by mating which normally resulted in fertilization and subsequent embryonic diapause. Renewed blastocyst development was initiated by removal or loss of a pouch young and birth followed about 27 days later.
Unlike other macropodids with a similar breeding pattern, birth, as a result of renewed blastocyst development near the end of a large young's pouch life, did not occur within a day or two of the permanent emergence of the young, but followed 16 to 29 days later. In M. r. rufogriseus , young that left the pouch permanently in the non-breeding period were not replaced by new young until the beginning of the next breeding season two to four months later, and blastocysts resulting from mating of females without pouch young at the end of the breeding season remained quiescent until the next breeding season five to eight months later.
Females of both subspecies first mated at an age of about 14 months, and males were producing mature spermatozoa by about 19 months.
Young first left the pouch for short periods at about 230 days of age and permanently at about 280 days.
Observations are also given on reproductive behaviour, interpretation of vaginal smears, sex ratio of young, selection of teat by pouch young, and development of morphological features in known-age young that may be used as an aid in age determination.     

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.     

The tammar wallaby: a model to examine endocrine and local control of lactation

Marsupials, such as the tammar wallaby (Macropus eugenii), have adopted a reproductive strategy that is very different to eutherians. Both the rate of production and the composition of milk changes progressively during the lactation cycle to meet the nutritional demands of an altricial young. The tammar therefore provides a valuable model to study changes in milk composition, and in particular the genes that code for proteins secreted in the milk, to more accurately assess the role of gene products regulating either development of the young or mammary function.     

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 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.     

Intestinal neuraminidase activity of suckling rats and other mammals. Relationship to the sialic acid content of milk.

1. The neuraminidase activity of homogenates of the mucosa of the middle and distal thirds of the small intestine of rats increased about 5-fold between birth and 4 to 8 days of age, and then gradually declined to the much lower adult activity by 24 days. No comparable changes occurred in the proximal third. 2. In 8-day-old rats, the neuraminidase activity of the middle and distal thirds of the small intestine was about 10 times greater than that of the proximal third, 20 times greater than that of the colon and at least 100 times greater than that of the liver, brain, gastric mucosa or pancreas. 3. In all other species investigated (mice, rabbits, cats and guinea pigs), the neuraminidase activity of the middle and distal thirds of the small intestine was greater in suckling animals than in adults. 4. The sialic acid content of rat milk increased about 2-fold between birth and 8 days post partum and then declined. 5. There was a highly significant positive correlation between the intestinal neuraminidase activity of suckling animals of various species and ages and the sialic acid content of milk obtained from the corresponding species and stage of lactation. 6. It is suggested that the intestinal neuraminidase of suckling mammals functions primarily to remove sialic acid from various components of milk, thus providing sialic acid for the synthesis of sialoglycoproteins and gangliosides by the young.