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.     

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.     

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.     

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 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.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.     

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii)

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.     

Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii)

Lactating tammars can provide two different milks simultaneously from adjacent glands to a young newborn (phase 2 of lactation) and an older animal at heel (phase 3 of lactation). Evidence that the two glands are controlled independently is shown by the capacity of mammary explants from these glands to synthesize different whey proteins and DNA and RNA at different rates. Prolactin is essential for the maintenance of milk synthesis, but its role in differential responses of the individual mammary glands remains to be established. Potential mechanisms for the control of milk synthesis are discussed.     

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 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.     

Development of urine concentrating ability in pouch young of a marsupial,the tammar wallaby (Macropus eugenii)

Summary During the first 28–30 weeks after birth, pouch young of the tammar wallaby (Macropus eugenii) normally produce urine less than 500 mOsm/kg and elevate their urine concentration by less than 20% when dehydrated by about 10% of body weight. The adult tammar, in contrast, can produce urine in excess of 3,000 mOsm/kg. The aim of this study was to determine when the various processes involved in urine concentration become mature in the tammar.Vasopressin was detectable in the pituitary of week-old tammars and pituitary vasopressin content decreased significantly after dehydration. Plasma vasopressin did not vary with age and dehydration was associated with an increase in plasma vasopressin levels. By 15 weeks of age at least, tammar kidney slices were able to bind vasopressin as indicated by a rise in tissue cAMP level following hormone treatment.The sodium and urea content of the renal medulla increased with age and significant gradients of these solutes were established by 25 weeks of age. Pouch young older than 25 weeks showed increased medullary sodium and urea levels following dehydration.The inability of pouch young less than 20 weeks of age to produce a highly concentrated urine does not result from any inadequacy in perception of osmotic stimuli or release of vasopressin by the pituitary or of binding of hormone by the kidney. Rather, it appears to be largely attributable to an insufficient medullary hypertonicity, particularly with respect to urea, which is consequent upon structural immaturity of the loop of Henle.Abbreviations cAMP cyclic AMP adenosine 3,5-monophosphate - AVP arginine vasopressin - LVP lysine vasopressin     

Changes in alpha-lactalbumin, total lactose, UDP-galactose hydrolase and other factors in tammar wallaby (Macropus eugenii) milk during lactation

alpha-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 weeks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The alpha-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free lactose plus lactose contained in oligosaccharides) fell to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDP-galactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid beta-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also play a role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid beta-galactosidase during or after secretion.     

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 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.     

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.     

Cyclic AMP-dependent tyrosine phosphorylation in tammar wallaby (Macropus eugenii) spermatozoa

Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues were principally located along the tammar sperm flagellum, and occasionally at a small region of the sperm head, adjacent to the acrosome. Associated with the tyrosine phosphorylation of tammar spermatozoa, was a change in sperm head conformation to a T-shaped orientation, further implying the importance of these pathways to normal tammar sperm function. Redox activity, as detected by lucigenin-dependent chemiluminescence, was stimulated by NADPH in caudal sperm preparations but not ejaculated spermatozoa. However, neither sperm population responded to treatment with NADPH with changes in intracellular cAMP or tyrosine phosphorylation. In conclusion, tammar spermatozoa possess the same cAMP-mediated, tyrosine phosphorylation-dependent signal transduction cascade that has been associated with capacitation in eutherian spermatozoa. However in Metatherian spermatozoa we could find no evidence that this pathway was redox regulated.     

Chromosomal localization of the gene for late lactation protein (LLP) in the tammar wallaby (Macropus eugenii)

The gene coding for the late lactation protein (LLP) in Macropus eugenii has been mapped using in situ hybridization to the proximal region of the long arm of chromosome 3. This position was confirmed by means of exclusion Southern blot analysis.     

A lactational study of the composition and integrity of casein micelles from the milk of the tammar wallaby (Macropus eugenii)

The amount of casein found in the milk of the tammar wallaby increases as lactation progresses. The increase is due to increasing amounts of β-casein; the α-casein remains largely constant. The α-casein is the more highly phosphorylated; the most abundant form is the 10-P, throughout lactation. The level of phosphorylation of β-casein shifts to lower average values in late lactation, possibly indicating the enzymatic reaction is overloaded by the increasing amounts of β-casein. Unlike bovine casein micelles, the wallaby micelles are not completely disrupted at pH 7.0 by sequestration of their calcium content with ethylene diamine tetraacetic acid (EDTA). Complete disruption only follows the addition of sodium dodecyl sulphate, indicating considerably greater importance for hydrophobic bonds in maintaining their integrity. This micellar behaviour indicates that, despite the evolutionary divergence of marsupials millennia ago, the caseins of wallaby milk assemble into micelles in much the same fashion as in bovine milk.