Old and new stories: revelations from functional analysis of the bovine mammary transcriptome during the lactation cycle

Abstract

The cow mammary transcriptome was explored at -30, -15, 1, 15, 30, 60, 120, 240, and 300 d relative to parturition. A total of 6,382 differentially expressed genes (DEG) at a false discovery rate ≤ 0.001 were found throughout lactation. The greatest number of DEG (>3,500 DEG) was observed at 60 and 120 d vs. -30 d with the largest change between consecutive time points observed at -15 vs. 1 d and 120 vs. 240 d. Functional analysis of microarray data was performed using the Dynamic Impact Approach (DIA). The DIA analysis of KEGG pathways uncovered as the most impacted and induced 'Galactose metabolism', 'Glycosylphosphatidylinositol (GPI)-anchor biosynthesis', and 'PPAR signaling'; whereas, 'Antigen processing and presentation' was among the most inhibited. The integrated interpretation of the results suggested an overall increase in metabolism during lactation, particularly synthesis of carbohydrates and lipid. A marked degree of utilization of amino acids as energy source, an increase of protein export, and a decrease of the protein synthesis machinery as well cell cycle also were suggested by the DIA analysis. The DIA analysis of Gene Ontology and other databases uncovered an induction of Golgi apparatus and angiogenesis, and the inhibition of both immune cell activity/migration and chromosome modifications during lactation. All of the highly-impacted and activated functions during lactation were evidently activated at the onset of lactation and inhibited when milk production declined. The overall analysis indicated that the bovine mammary gland relies heavily on a coordinated transcriptional regulation to begin and end lactation. The functional analysis using DIA underscored the importance of genes associated with lactose synthesis, lipid metabolism, protein synthesis, Golgi, transport, cell cycle/death, epigenetic regulation, angiogenesis, and immune function during lactation.

Figure 1. Number of differentially expressed genes with FDR≤0.001 and post-hoc P<0.001 at each time relative to −30 d (pregnancy) (upper left panel), and relative to the previous time (bottom left panel).

A 2-fold (100%)-change threshold was applied for both the panel encompassing each time relative to −30 d (pregnancy) (upper right panel) and the comparison of each time relative to the previous time point (bottom right panel). Applying such a threshold highlighted the increase in the proportion of up-regulated genes within DEG that were markedly affected. Additional images with several thresholds for each type of comparison are available in Figure S2).

Figure 2. Overall view of the DIA results of the KEGG pathways with main categories and subcategories as provided by KEGG (http://www.genome.jp/kegg/pathway.html).

Reported are all times compared with −30 d, and the two consecutive times (1 vs. −15 d and 240 vs. 120 d) with the largest number of differentially expressed genes and overall impact on pathways. Blue horizontal bars denote the impact and the square on their right denote the direction of the impact (red = activation; green = inhibition). See companion paper for interpretation of the DIA output.

Figure 3. TreeMap visualization obtained by REVIGO analysis (see Materials and Methods for details) of the summary of GO Biological process deemed by DIA to be overall activated during full lactation (from 15 to 120 d) compared to −30 d.

Similar colors denote semantic similarity and dimension of the area is proportional to the overall direction of the impact, as calculated by the DIA. Not all the terms are reported due to space constraints (see Additional file S4 for details).

Figure 4. TreeMap visualization obtained by REVIGO analysis (see Materials and Methods for details) of the summary of GO Biological process deemed by DIA to be overall inhibited during full lactation (from 15 to 120 d) compared to −30 d.

Similar colors denote semantic similarity and dimension of the area is proportional to the overall direction of the impact, as calculated by the DIA. Not all the terms are reported due to space constraints (see Additional file S4 for details).

Figure 5. Summary figure of the most relevant biological functions in bovine mammary gland during the lactation cycle as revealed by the Dynamic Impact Approach (DIA) analysis of KEGG pathways and Gene Ontology terms (see Additional file S1 for details).

Shown at the center are the curve of lactation, yield of main milk constituents (protein, lactose, and fat) , , and estimates of fatty acid import and de novo synthesis . Red titles are the most relevant metabolism as revealed by the KEGG pathway and Gene Ontology analysis. Other color titles denote main functions impacted during lactation. The large increase at the transcriptomic level of caseins or other milk proteins are not shown. The X-axis depicts the day relative to parturition and the Y-axis the direction of the impact as calculated by DIA. Legend: AA = amino acid; Arg = arginine; Ca = calcium; Chol = cholesterol; Cl = chloride; Cu = copper; Cys = cysteine; ER = endoplasmic reticulum; FA = fatty acids; GnRH = Gonadotropin-releasing hormone pathway; Gly = glycine; GPI = Glycophosphatidylinositol; His = histidine; Ile = isoleucine; LCFA = long-chain fatty acids; Leu = leucine; Met = methionine; mTOR = mTOR = mammalian target of rapamycin signaling pathway; Na = sodium; NH4 = ammonium; Notch = Notch signaling pathway; P = phosphate; PPAR = peroxisome proliferator-activated receptor signaling pathway; S = sulfate; Ser = serine; TAG = triacylglycerol; Thr = threonine; Val = valine; VEGF = vascular endothelial growth factor signaling pathway.