Project description:During lactation, mammary epithelial secretory cells secrete huge amounts of milk from their apical side. The major milk proteins, the caseins, are secreted by exocytosis, while milk fat globules are released by budding, enwrapped by the plasma membrane. Due to the number and large size of milk fat globules, the membrane surface needed for their secretion might exceed that of the apical plasma membrane. In order to identify the cellular compartments that may provide membrane during the budding of milk fat globules, a large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globules membranes was performed in mouse cells. The differential analysis of the protein profiles of these two organelles strongly suggest that, in addition to the apical plasma membrane, at least the endoplasmic reticulum, the secretory vesicles containing caseins, and potentially the mitochondria contribute to the formation of the milk fat globule membrane. Moreover, the specific analysis of the membrane-associated as well as the raft-associated proteins reinforces this possibility and points to a role for lipid rafts in milk product secretion. The subcellular localization of several major proteins of the cytoplasmic lipid droplets or of the milk fat globule membrane was investigated by immunofluorescence in lactating mammary epithelial cells. Furthermore, the localization of GM1 ganglioside, a known marker of lipid rafts and of free cholesterol, showed a clear association with both cytoplasmic lipid droplets and secreted milk fat globules. Additionally, the presence of some SNARE proteins that may be involved in casein exocytosis and of two Rab GTPases on both cytoplasmic lipid droplets and milk fat globules was demonstrated by immunofluorescence. Altogether, our results provide evidence for a pivotal role of the endoplasmic reticulum as membrane contributor to milk fat globule budding, and suggest that some SNARE proteins may spatio-temporally coordinate the secretion of milk products.

Project description:Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, the transcriptome derived from the milk fat layer has not been compared with the mammary-derived transcriptome nor have sources of RNA been quantified in milk. In this study the effects of milk collection and processing on RNA quality and origin were assessed in humans and rhesus macaques. Total RNA in milk was quantitated in acridine orange-stained milk using an automated whole slide scanner and custom-built Globulator software. Total RNA extracted from milk fat, cells in milk, and mammary biopsies of lactating rhesus macaques were compared using RNA sequencing and analysis. Compared with human milk, milk from macaques contained similar amounts of RNA-containing cytoplasmic crescents, but more cells. Total RNA extracted from milk fractions was also evaluated for factors that affect RNA quality. Degradation of RNA extracted from human milk fat was positively correlated with geographic distance from collection site, storage time, and sample type. There were no differences in RNA degradation in macaque milk collected after 10 min or 4 hr accumulation, suggesting that degradation of RNA extracted from milk fat may not occur in the mammary gland. Using RNA-Seq, RNA extracted from macaque milk fat and cells in milk more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from mammary epithelial cells. Analysis of highly abundant putative microRNAs in macaque milk fat revealed a potentially novel non-coding RNA species that is conserved in humans. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells. However, this sample type clearly requires protocols that minimize RNA degradation. Transcript profiles from milk cells, milk fat, and mammary tissue from 6 lactating rhesus macaques at 30 and 90 days lactation; 34 samples run in triplicate

Project description:Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, the transcriptome derived from the milk fat layer has not been compared with the mammary-derived transcriptome nor have sources of RNA been quantified in milk. In this study the effects of milk collection and processing on RNA quality and origin were assessed in humans and rhesus macaques. Total RNA in milk was quantitated in acridine orange-stained milk using an automated whole slide scanner and custom-built Globulator software. Total RNA extracted from milk fat, cells in milk, and mammary biopsies of lactating rhesus macaques were compared using RNA sequencing and analysis. Compared with human milk, milk from macaques contained similar amounts of RNA-containing cytoplasmic crescents, but more cells. Total RNA extracted from milk fractions was also evaluated for factors that affect RNA quality. Degradation of RNA extracted from human milk fat was positively correlated with geographic distance from collection site, storage time, and sample type. There were no differences in RNA degradation in macaque milk collected after 10 min or 4 hr accumulation, suggesting that degradation of RNA extracted from milk fat may not occur in the mammary gland. Using RNA-Seq, RNA extracted from macaque milk fat and cells in milk more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from mammary epithelial cells. Analysis of highly abundant putative microRNAs in macaque milk fat revealed a potentially novel non-coding RNA species that is conserved in humans. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells. However, this sample type clearly requires protocols that minimize RNA degradation.

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Homo sapiens

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