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

The 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.

The fur seal — a model lactation phenotype to explore molecular factors involved in the initiation of apoptosis at involution

Mammary gland involution requires co-ordination of milk production, immune responses, apoptosis and remodeling. Initiation and progression of each of these components involves integral control by the mammary gland. Although cell-based culture models and genetically manipulated animals have shed light on these processes, the factors controlling each step in the involution cascade are still poorly understood. The fur seal displays a unique lactation phenotype. During the lactation cycle the mammary gland downregulates milk production and initiates an immune response but fails to initiate the apoptotic phase of involution, allowing the female fur seal to undertake long foraging trips of up to 28 days between suckling bouts. Upon return to shore the female continues feeding her pup following resumption of lactation and milk production. Expression profiling of genes involved in this lactation cycle provides valuable tools for investigation of the factors responsible for the initiation of apoptosis at involution.

Fur seal mammary gland evasion of involution and cell-matrix interactions

This thesis aimed to exploit the Cape fur seal as an alternative model to study mammary gland development and especially the switch from lactation to involution, as well as better understand cell-matrix interactions in vitro, essential to understanding tissue homeostasis and pathogenesis.

The relationship between milk, mammary adipocytes and ECM in regulating murine mammary gland involution

 This thesis investigated the role of milk, extracellular matrix and mammary adipocytes in regulating mammary gland function during involution in mice and explored the use of an in vitro culture model, the mammosphere model system to study the same.

Analysis of human breast milk cells: gene expression profiles during pregnancy, lactation, involution, and mastitic infection

The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step toward understanding why some women display poor lactation outcomes. Here, we demonstrate the utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from five time points (24 h prepartum during the colostrum period, midlactation, two involutions, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (e.g., CEL, OLAH, FOLR1, BTN1A1, and ARG2), while milk cells collected during involution showed a significant downregulation of milk synthesis genes and activation of involution associated genes (e.g., STAT3, NF-kB, IRF5, and IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, while maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MECs within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland.

Fur seal adaptations to lactation: insights into mammary gland function

The fur seal (Arctocephalus spp. and Callorhinus spp., members of the pinniped family) is a mammal with the unusual capability to modulate its lactation cycle by turning milk production on and off without the typical mammalian regression and involution of the mammary gland. Lactation has evolved from constraints arising from the spatial and temporal separation of infant nursing and maternal foraging as the mother gives birth and feeds the pup on land while acquisition of nutrients for milk production occurs at sea. The lactation cycle begins with the female fur seal undergoing a perinatal fast of approximately 1 wk, after which time she departs the breeding colony to forage at sea. For the remainder of the long lactation period (116–540 days), the mother alternates between short periods ashore suckling the young with longer periods of up to 4 wk of foraging at sea. Milk production continues while foraging at sea, but at less than 20% the rate of production on land. Fur seals produce one of the richest milk reported, with a very high lipid content contributing up to 85% of total energy. This feature serves as an adaptation to the young's need to produce an insulating blubber layer against heat loss and to serve as an energy store when the mother is away foraging at sea. This atypical pattern of lactation means mothers have long periods with no suckling stimulus and can transfer high-energy milk rapidly while on land to minimize time away from foraging grounds. The absence of suckling stimulus and milk removal during foraging does not result in the onset of involution with associated apoptosis of mammary secretory cells and a subsequent progressive breakdown of the cellular structure of the mammary gland. The mechanisms controlling lactation in the fur seal mammary gland have been investigated using molecular and cellular techniques. These findings have shed light on the processes by which the unique features of lactation in the fur seal are regulated.

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10–12 month lactation period, female fur seals suckle pups on shore for 2–3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 μm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (αS2-casein, β-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell–matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Inflammatory pseudotumour of the liver : the residuum of a biliary cystadenoma?

Inflammatory pseudotumour is a rare form of a liver mass. We report the case of a 28-year-old man presenting with obstructive jaundice, in whom an inflammatory pseudotumour arose with the resolution of a mucus secreting cystic liver lesion. The initial features suggested an intrahepatic cystadenoma or cystadenocarcinoma, which on its involution left a solid mass. Histopathology showed an inflammatory pseudotumour with no evidence of malignancy. A similar case has been reported recently, with the development of an inflammatory pseudotumour following collapse of a liver cyst seen on imaging. These two cases may shed some light on the origins of these rare liver lesions.

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.

Conservation of the ST6Gal I gene and its expression in the mammary gland

Milk sialoglycoconjugates can protect the gastrointestinal tract of the suckling neonate by competitively binding to invading pathogens and promoting growth of beneficial flora, and their potential role in postnatal brain development is of particular interest in human infant nutrition. Although the concentration and the distribution of sialoglycoconjugates have been extensively studied in the milk of various species, the investigation of sialyltransferase gene expression in the mammary gland, in the context of lactation, has been limited. The sialyltransferase enzyme ST6Gal I transfers sialic acid from CMP-sialic acid to type 2 (Galβ1,4GlcNAc) free disaccharides or the termini of N- or O-linked oligosaccharides using an α2,6-linkage. Expression of the ST6Gal I gene is primarily regulated at the level of transcription through the use of several cell and development- specific promoters, producing transcripts with divergent 5′ untranslated regions (UTR). In the mouse mammary gland, the novel 5′UTR exon (L) appears to be associated with a drastic increase in ST6Gal I gene expression during lactation. We find that rats also possess an exon (L), suggesting conservation of this regulatory mechanism in rodents. In contrast, an exon (L)-containing transcript was not detected in the lactating bovine or human mammary gland. We also observed a trend of increasing ST6Gal I gene expression in the bovine mammary gland, culminating in involution. This is in contrast to species such as mice where the greatest change in ST6Gal I gene expression occurs between pregnancy and lactation, suggesting different roles in rodents vs. other mammals for α2,6-sialylated oligosaccharides present in milk.

Resistive exercises, with or without whole body vibration, prevent vertebral marrow fat accumulation during 60 days of head-down tilt bed rest in men

Fat accumulates in the bone marrow of lumbar vertebrae with bed rest. Exercise with or without whole body vibration may counter this effect. Our objectives were to measure 1) the vertebral fat fraction (VFF) of men subjected to bed rest who performed resistive exercises with (RVE, n = 7) or without whole body vibration(RE, n = 8) or no exercise (CTR, n = 9) using three MRI techniques; and 2) changes in peripheral blood counts. Twenty-four healthy men (age: 20-45 yr) underwent -6° head-down tilt (HDT) bed rest for 60 days. MRI was performed using three techniques (fat saturation, proton spectroscopy, and in and out of phase) to measure the fat fraction of L(3), L(4), and/or L(5) at baseline, mid-HDT, and end-HDT. Erythrocytes and leukocytes were counted at HDT days 19, 33, 47, 54, and 60. The mean absolute VFF was increased in the CTR group at mid-HDT and end-HDT (+3.9 ± 1.3 and +3.6 ± 1.2%, respectively, both P < 0.05). The RE group had a smaller VFF change than the CTR group at mid-HDT (-0.9 ± 1.2 vs. +3.9 ± 1.3%, P < 0.05). The RVE group had a smaller VFF change than the CTR group at end-HDT (-2.6 ± 1.9 vs. +3.5 ± 1.2%, P < 0.05). Erythrocyte counts were increased in all groups at HDT day 19 and HDT day 33 and in the RE group at HDT day 54 (all P < 0.05). Bed rest for 60 days at -6° HDT increased lumbar VFF in men beyond natural involution. RVE and RE regimens effectively prevented VFF accumulation. Higher erythrocyte counts were not altered by RVE or RE. Whole body vibration, along with RE administered to people with prolonged immobility, may prevent fat accumulation in their bone marrow.