Molecular evolution of a novel marsupial S100 protein (S100A19) which is expressed at specific stages of mammary gland and gut development.

S100 proteins are calcium-binding proteins involved in controlling diverse intracellular and extracellular processes such as cell growth, differentiation, and antimicrobial function. We recently identified a S100-like cDNA from the tammar wallaby (Macropus eugenii) stomach. Phylogentic analysis shows wallaby S100A19 forms a new clade with other marsupial and monotreme S100A19, while this group shows similarity to eutherian S100A7 and S100A15 genes. This is also supported by amino acid and domain comparisons. We show S100A19 is developmentally-regulated in the tammar wallaby gut by demonstrating the gene is expressed in the forestomach of young animals at a time when the diet consists of only milk, but is absent in older animals when the diet is supplemented with herbage. During this transition the forestomach phenotype changes from a gastric stomach into a fermentation sac and intestinal flora changes with diet. We also show that S100A19 is expressed in the mammary gland of the tammar wallaby only during specific stages of lactation; the gene is up-regulated during pregnancy and involution and not expressed during the milk production phase of lactation. Comparison of the tammar wallaby S100A19 protein sequence with S100 protein sequences from eutherian, monotreme and other marsupial species suggest the marsupial S100A19 has two functional EF hand domains, and an extended His tail. An evolutionary analysis of S100 family proteins was carried out to gain a better understanding of the relationship between the S100 family member functions. We propose that S100A19 gene/protein is the ancestor of the eutherian S100A7 gene/protein, which has subsequently modified its original function in eutherians. This modified function may have arisen due to differentiation of evolutionary pressures placed on gut and mammary gland developmental during mammal evolution. The highly regulated differential expression patterns of S100A19 in the tammar wallaby suggests that S100A19 may play a role in gut development, which differs between metatherians and eutherians, and/or include a potential antibacterial role in order to establish the correct flora and protect against spiral bacteria in the immature forestomach. In the mammary gland it may protect the tissue from infection at times of vulnerability during the lactation cycle.

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.

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.