Biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two galactosyltransferases in lactating mammary glands of the tammar wallaby, Macropus eugenii

Tammar wallaby (Macropus eugenii) mammary glands contain two galactosyltransferases of which the first, 4 beta GalT, is a UDP-galactose:N-acetylglucosaminyl beta 1----4-galactosyltransferase equivalent to the A protein of the lactose synthase of eutherian mammals. The second enzyme, 3 beta GalT, is a UDP-galactose:lactose beta 1----3-galactosyltransferase, not previously identified in mammary glands of any species, which catalyses the formation of Gal beta 1----3 Gal beta 1----4 Glc from lactose. The two enzyme activities, as well as the lactose synthase activity, have been characterised with respect to the effects of pH, apparent Km values, effects of bovine and tammar alpha-lactalbumins, heat sensitivity and identity of products. Studies on the substrate specificity and heat sensitivity of the 3 beta GalT activity suggest that this enzyme may catalyse the beta-galactosylation of Gal beta 1----3Gal beta 1----4Glc as well as of lactose. The activity of the 3 beta GalT, unlike that of the 4 beta GalT, changes dramatically during the course of lactation in parallel with similar changes in the carbohydrate content of tammar milk.     

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.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Immunological aspects of gestation in the tammar wallaby, Macropus eugenii.

Sensitization to male histocompatibility antigens and repeated pregnancy to the same male were found to have little effect on fertility or length of gestation in the tammar wallaby, M. eugenii. However, in some sensitized females a long interval occurred between removal of pouch young and the next birth. In addition to studies on fertility, the immunological response of female tammars to their mate has been examined by one-way mixed leucocyte culture (MLC) carried out at the beginning and end of one breeding season. In virgin females, examined at the beginning of the breeding season, the MLC response to the prospective mate peaked on day 6. In contrast, by the end of the season, MLC responses were much lower and peaked earlier, on day 3 to day 5.     

The first comprehensive genetic linkage map of a marsupial: the tammar wallaby (Macropus eugenii)

The production of a marsupial genetic linkage map is perhaps one of the most important objectives in marsupial research. This study used a total of 353 informative meioses and 64 genetic markers to construct a framework genetic linkage map for the tammar wallaby (Macropus eugenii). Nearly all markers (93.8%) formed a significant linkage (LOD > 3.0) with at least one other marker, indicating that the majority of the genome had been mapped. In fact, when compared with chiasmata data, >70% (828 cM) of the genome has been covered. Nine linkage groups were identified, with all but one (LG7; X-linked) allocated to the autosomes. These groups ranged in size from 15.7 to 176.5 cM and have an average distance of 16.2 cM between adjacent markers. Of the autosomal linkage groups (LGs), LG2 and LG3 were assigned to chromosome 1 and LG4 localized to chromosome 3 on the basis of physical localization of genes. Significant sex-specific distortions toward reduced female recombination rates were revealed in 22% of comparisons. When comparing the X chromosome data to closely related species it is apparent that they are conserved in both synteny and gene order.     

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.     

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     

PCR derived cDNA clones for X-linked phosphoglycerate kinase-1 in a marsupial, the tammar wallaby (Macropus eugenii).

cDNA clones for the X-linked PGK-1 were obtained from a tammar wallaby liver by PCR and sequenced. The PGK-1 gene published here is the consensus sequence of those clones. The sequence represents an open reading frame of 1251 bp. Sequence comparisons to X-linked and autosomal sequences showed the greatest homology with the X-linked PGK-1 genes in eutherian species. This sequence opens the way for studying the paternal X inactivation phenomenon in marsupials and will assist in defining the time course of mammalian evolution.     

Lactation in the tammar wallaby (Macropus eugenii). II. Intake of milk components and maternal allocation of energy

The intake of milk components (total solids, carbohydrate, protein, lipid, energy) by suckling tammar wallabies ( Macropus eugenii ), from peak lactation to independence, was measured using a double-isotope dilution technique and chemical analysis of milk samples. The time of peak intake of milk solids (day 256 of lactation or 1126 g of offspring weight) was similar to that for whole milk. Peak intake of carbohydrate occurred earlier than this (235 days) and peak intakes of protein and lipid occurred later (262 days and 266 days, respectively). Intake of gross energy peaked at 262 days and represented a maternal yield of about 207 KJ.Kg^-0.75.d^-1. This is much lower than peak lactational energy yields in most other mammals, but the duration of lactation is longer in tammars than in other mammals. Total output of energy in milk by tammar mothers was 63 MJ, and this would require an intake of about 98 MJ of metabolizable energy in food. This requirement, which is equivalent to 21 MJ/kg of maternal weight, is similar to those calculated for sheep and cattle, suggesting that there are not large differences between marsupial and placental herbivores in terms of weight-related allocation of energy to reproduction. We suggest that a strategy of minimizing the peak energetic demand of lactation may be an important adaptation for a small, primarily grazing mammal due to size-related physiological constraints on elevating herbage intake and the increasing risk from predators when grazing time is increased.     

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.     

Ultrastructural localization of relaxin in the corpus luteum of the pregnant and early lactating tammar wallaby, Macropus eugenii

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200–350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition. Received: 3 March 1997 / Accepted: 26 May 1997     

Characterization and expression of Peroxiredoxin 1 in the neonatal tammar wallaby (Macropus eugenii)

Peroxiredoxin 1 (PRDX1) is a ubiquitously expressed antioxidant with vital roles in basal metabolic functions. In addition PRDX1 is involved in cell differentiation and proliferation, apoptosis and innate immunity. In this study, we have characterized PRDX1 from the tammar wallaby (Macropus eugenii). Tammar PRDX1 has high conservation of functional residues and motifs, and demonstrates a close homology with eutherian and vertebrate orthologues. Stimulation of adult tammar leukocytes with lipopolysaccharide and lipoteichoic acid suggests a role for PRDX1 in innate immune defences. PRDX1 expression in the organs of tammar pouch young was mildly elevated early in life possibly reflecting its role in basal metabolic processes. Later increases in PRDX1 expression correlated with functional maturation of several immune organs or with preparation for increased oxidative stress of emergence. The findings of the study are reflections of the complex integrated roles that PRDX1 has in regulation of oxidative stress, apoptosis, cell differentiation and proliferation, and innate immunity.     

Uterine and embryonic metabolism after diapause in the tammar wallaby, Macropus eugenii

Pouch young were removed from lactating tammars to terminate embryonic diapause. Uterine metabolism was assessed at periods afterwards by incubating endometrial explants with [3H]leucine, and measuring the incorporation into acid-soluble material. Blastocysts were incubated with [3H]uridine to assess uptake and incorporation into acid-soluble material. Uterine reactivation, shown by an increase in the rate of leucine incorporation into secreted protein, was evident by Day 4 after removal of pouch young and was significantly more in both secreted and tissue protein by Day 6. Both continued to increase in gravid and non-gravid uteri up to Day 12. By the end of pregnancy (Day 26) uterine metabolism in the gravid uterus produced 2-3 times more secreted protein than in the non-gravid uterus, demonstrating a local feto-placental influence on the uterus. Tissue incorporation had declined in endometrium of gravid and non-gravid uteri by Day 26. Day 5 embryos were metabolically more active than in quiescence, although expansion of the embryos was not seen until Day 9. The early reactivation of the uterus and embryo from diapause suggests that it is not triggered by the previously described peaks of progesterone and oestradiol in plasma at Day 5, although there may be an earlier, increased sensitivity to these steroids which allows uterine reactivation to precede changes in peripheral plasma concentration.     

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.     

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.