Sequencing the transcriptome of milk production: milk trumps mammary tissue

Abstract

Background: 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, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells.

Results: We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole 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 milk-producing mammary epithelial cells.

Conclusions: RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

Figures

Figure 1

Composite images of whole milk stained with acridine orange (AO). Each image contains three channels: 1) a differential interference contrast image to view the fat globules, which look like gray bubbles; 2) a fluorescence channel for AO-RNA; and 3) another fluorescence channel for AO-DNA. When AO associates with RNA, the emission maximum is 650 nm (red). When AO associates with DNA, the emission maximum is 525 nm (green). Therefore, areas containing only RNA (e.g. crescents) look red, only DNA look green, and both RNA and DNA (e.g. nucleated cells) look yellow. (A) Close-up of human whole milk with a nucleated cell (yellow) and several crescents of various sizes. (B) Whole slide scanned image of human whole milk collected after a 4-hr milk accumulation. (C–D) Whole slide scanned images of macaque whole milk after (C) a 4-hr and (D) a 10-min milk accumulation.

Figure 2

Percentage of cytoplasmic crescents and cells in milk. Milk samples were collected after a 4-hr accumulation period in human subjects and a 4-hr, 2-hr, or 10-min accumulation in macaques. The boxplots show the (A) percentage of milk fat globules with cytoplasmic crescents and (B) the percentage of RNA attributable to nucleated cells in these milk samples. (A-B) The upper and lower bounds of each box denote the first and third quartiles of the data, the dark horizontal line within each box denotes the median value, and the whiskers extend to the minimum and maximum values. The width of each box is proportional to the square root of the samples size. Asterisks denote statistical significance (p < = 0.05).

Figure 3

Effect of milk fraction and processing time on RNA integrity. (A-B) RNA degradation by sample types in (A) human and (B) macaque samples. All differences are significant (t-test, p < = 0.05). (C-D) Human milk fat RNA degradation by processing time in terms of (C) the distance to the lab from the site of collection (R2 = 0.40, p = 0.012) and (D) the amount of storage time in TRIzol at -80°C prior to RNA isolation (R2 = 0.26, p = 0.044).

Figure 4

Hematoxylin- and eosin-stained mammary biopsy samples from rhesus macaques. Histology of tissue biopsied from the mammary glands of rhesus macaques at different stages of lactation. Sections (4 μm) were stained with hematoxylin and eosin. Scale bar = 100 μm. (A) Multiparous aged female macaque, non-pregnant, non-lactating; (B) multiparous female macaque, lactation day 30; and (C) multiparous female macaque, lactation day 90.

Figure 5

Expression of cell-specific markers in milk and mammary transcriptomes. Each box in the heatmap represents log-transformed expression intensity. The color key indicates the level of expression: Green = low/no expression, Yellow = low expression, Orange = moderate expression, Pink = high expression, and White = very high expression. The color key also includes a light blue line that indicates the number of observations at each level of expression.

Figure 6

Abundances of transcripts that code for proteins in the lactose synthesis pathway. All differences between mammary biopsy (“Bx”) and milk fat samples (“Fat”) were significant. Differences between milk fat (“Fat”) and milk cell (“Cells”) samples were significant for all transcripts except LALBA and B4GALT.

Figure 7

Transcriptome homogeneity within sample types. Transcriptomes derived from samples of the same type (mammary biopsy, milk cells, or milk fat) were compared with other samples of the same type using a Spearman’s correlation. The boxplot summarizes the findings by sample type. The upper and lower bounds of each box denote the first and third quartiles of the data, the dark horizontal line within each box denotes the median value, and the whiskers extend to the minimum and maximum values. The width of each box is proportional to the square root of the samples size. Asterisks denote statistical significance (p < = 0.05).

Figure 8

Correlation of milk and mammary transcriptomes. Each square in the heatmap represents the pairwise correlation of the sample listed in the row compared with the sample listed in the column. The shades of blue across the top of each heatmap correspond to sample type. The colors on the left side of each heat map correspond to each individual monkey. Both biological and technical replicates are shown. All technical replicates appear as clustered triplets in the dendograms. (A) Milk fat vs mammary biopsy samples; dark blue denotes biopsy, light blue denotes milk fat. (B) Milk cells vs mammary biopsy samples, dark blue denotes milk cell, light blue denotes milk fat. (C) Milk cells vs milk fat samples, dark blue denotes milk cell, blue denotes biopsy. (D) Mammary biopsy samples at day 30 and day 90. (E) Milk fat samples at day 30 and day 90. (F) Milk cells samples at day 30 and day 90. (D–F) Dark and light blue distinguish day 30 and 90 samples.