Kevin R. Nicholas

The tammar wallaby : A model to study putative autocrine-induced changes in milk composition

The marsupial newborn is immature and the mother has the capacity to alter milk composition significantly during lactation, presumably to meet the nutritional requirements of the developing young. Furthermore, macropodid marsupials may practice asynchronous concurrent lactation (ACL)7whereby the mother provides milk which differs in all the major components from adjacent mammary glands for two young of different ages. This phenomenon suggests that local regulation of mammary function, in addition to endocrine stimuli, is likely to be important for controlling milk composition. This paper explores the possibility that changes in sucking patterns of the young represent the first step in a mechanism to signal the mammary gland for putative autocrine-induced changes in milk composition.

Maternal Regulation of Milk Composition, Milk Production, and Pouch Young Development During Lactation in the Tammar Wallaby (Macropus eugenii )

Abstract 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.

Evolution of Lactation: Ancient Origin and Extreme Adaptations of the Lactation System

Lactation, an important characteristic of mammalian reproduction, has evolved by exploiting a diversity of strategies across mammals. Comparative genomics and transcriptomics experiments have now allowed a more in-depth analysis of the molecular evolution of lactation. Milk cell and mammary gland genomic studies have started to reveal conserved milk proteins and other components of the lactation system of monotreme, marsupial, and eutherian lineages. These analyses confirm the ancient origin of the lactation system and provide useful insight into the function of specific milk proteins in the control of lactation. These studies also illuminate the role of milk in the regulation of growth and development of the young beyond simple nutritive aspects.

The Gene for a Novel Member of the Whey Acidic Protein Family Encodes Three Four-disulfide Core Domains and Is Asynchronously Expressed during Lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of thetWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

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.

Insulin regulates milk protein synthesis at multiple levels in the bovine mammary gland

The role of insulin in milk protein synthesis is unresolved in the bovine mammary gland. This study examined the potential role of insulin in the presence of two lactogenic hormones, hydrocortisone and prolactin, in milk protein synthesis. Insulin was shown to stimulate milk protein gene expression, casein synthesis and 14C-lysine uptake in mammary explants from late pregnant cows. A global assessment of changes in gene expression in mammary explants in response to insulin was undertaken using Affymetrix microarray. The resulting data provided insight into the molecular mechanisms stimulated by insulin and showed that the hormone stimulated the expression of 28 genes directly involved in protein synthesis. These genes included the milk protein transcription factor, ELF5, translation factors, the folate metabolism genes, FOLR1 and MTHFR, as well as several genes encoding enzymes involved in catabolism of essential amino acids and biosynthesis of non-essential amino acids. These data show that insulin is not only essential for milk protein gene expression, but stimulates milk protein synthesis at multiple levels within bovine mammary epithelial cells.

Milk secretion in the rat : progressive changes in milk composition during lactation and weaning and the effect of diet

1. Progressive changes in the composition of milk from rats has been studied from day 0 to 20 of lactation and for 3 days following separation of the dams and pups at day 20 post partum. 2. The changes in concentration of Na, K and lactose suggested that secretion both prepartum and following weaning occurred by a paracellular mechanism whereas a transcellular pathway existed during established lactation. 3. The concentration of total protein and casein increased gradually throughout lactation. In contrast, the concentration of serum albumin increased and transferrin decreased markedly during early lactation. The fat content of milk declined 3-fold within 5 days of birth but the concentration of Ca, Mg and inorganic P increased. The concentration of each of these milk constituents remained constant during established lactation. 4. Following weaning the pronounced decline in lactose, K and inorganic P was negatively correlated with an increase in all other milk constituents except fat. 5. Rats fed a low energy diet produced milk with a lower fat content but with an unaltered concentration of protein and carbohydrate. The growth rate of these litters was similar for the first 5 days of lactation when compared to litters from dams fed a high energy diet. The growth rate of litters thereafter and following weaning was greater for rats fed a high energy diet.

Lactation transcriptomics in the Australian marsupial, Macropus eugenii: transcript sequencing and quantification

BackgroundLactation is an important aspect of mammalian biology and, amongst mammals, marsupials show one of the most complex lactation cycles. Marsupials, such as the tammar wallaby (Macropus eugenii) give birth to a relatively immature newborn and progressive changes in milk composition and milk production regulate early stage development of the young.ResultsIn order to investigate gene expression in the marsupial mammary gland during lactation, a comprehensive set of cDNA libraries was derived from lactating tissues throughout the lactation cycle of the tammar wallaby. A total of 14,837 express sequence tags were produced by cDNA sequencing. Sequence analysis and sequence assembly were used to construct a comprehensive catalogue of mammary transcripts.Sequence data from pregnant and early or late lactating specific cDNA libraries and, data from early or late lactation massively parallel sequencing strategies were combined to analyse the variation of milk protein gene expression during the lactation cycle.ConclusionResults show a steady increase in expression of genes coding for secreted protein during the lactation cycle that is associated with high proportion of transcripts coding for milk proteins. In addition, genes involved in immune function, translation and energy or anabolic metabolism are expressed across the lactation cycle. A number of potential new milk proteins or mammary gland remodelling markers, including noncoding RNAs have been identified.

Lack of functional alpha-lactalbumin prevents involution in Cape fur seals and identifies the protein as an apoptotic milk factor in mammary gland involution

BackgroundThe mammary gland undergoes a sophisticated programme of developmental changes during pregnancy/lactation. However, little is known about processes involving initiation of apoptosis at involution following weaning. We used fur seals as models to study the molecular process of involution as these animals display a unique mammary gland phenotype. Fur seals have long lactation periods whereby mothers cycle between secreting copious quantities of milk for 2 to 3 days suckling pups on land, with trips to sea alone to forage for up to 23 days during which time mammary glands remain active without initiating apoptosis/involution.ResultsWe show the molecular basis by which alpha-lactalbumin (LALBA), a secreted milk protein, is absent in Cape fur seals and demonstrate an apoptotic function for LALBA when exposed to mammary cells.ConclusionWe propose that apoptosis does not occur in fur seal mammary glands due to lack of LALBA in fur seal milk, allowing evasion of involution during a foraging trip. Our work identifies LALBA as a milk factor that feeds back on the mammary gland to regulate involution.

Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula).

The whey acidic protein (WAP) is a whey protein found in the milk of a number of species. We have isolated and characterised a WAP cDNA clone from the brushtail possum (Trichosurus vulpecula) and examined its expression in the mammary gland. The amino acid sequences of WAP from the possum and another marsupial, the tammar wallaby, share 69% identity, however, less sequence identity exists between the marsupial and eutherian WAP sequences (30-37%). The possum and tammar WAP genes consist of three four-disulphide core (4-DSC) domains, with a WAP motif at the beginning of each domain. In contrast, the eutherian WAP sequences consist of two 4-DSC domains with the WAP motif only present in the second domain. This WAP motif is also present in a number of protease inhibitors found in a wide range of species. Phylogenetic analysis of marsupial and eutherian WAP sequences suggests that the ancestral WAP gene has three domains and that one of the domains has been deleted from the eutherian gene. The profile of WAP gene expression in the possum mammary gland changed throughout lactation, with WAP mRNA levels reaching a peak between days 106 and 177 of lactation. The level of WAP mRNA in the mammary gland appeared to be correlated with the level of circulating prolactin in the lactating female and was different to that observed for several other whey protein genes. Overlapping expression of the WAP and early lactation protein genes, both of which are putative protease inhibitors, may provide protection of milk immunoglobulins that are required for the prolonged period of passive immune transfer to the marsupial pouch young.