Circulating exosomes may identify biomarkers for cows at risk for metabolic dysfunction

Abstract

Disease susceptibility of dairy cows is greatest during the transition from pregnancy to lactation. Circulating exosomes may provide biomarkers to detect at-risk cows to enhance health and productivity. From 490 cows, animals at high- (n = 20) or low-risk (n = 20) of transition-related diseases were identified using plasma non-esterified fatty acid and β-hydroxybutyrate concentrations and liver triacylglyceride concentrations during the two weeks post-calving. We isolated circulating exosomes from plasma of dairy cows at low-risk (LR-EXO) and high-risk (HR-EXO), and analyzed their proteome profiles to determine markers for metabolic dysfunction. We evaluated the effects of these exosomes on eicosanoid pathway expression by bovine endometrial stromal (bCSC) and epithelial (bEEL) cells. HR-EXO had significantly lower yield of circulating exosomes compared with LR-EXO, and unique proteins were identified in HR-EXO and LR-EXO. Exposure to LR-EXO or HR-EXO differentially regulated eicosanoid gene expression and production in bCSC and bEEL cells. In bCSC, LR-EXO exposure increased PGE₂ and PGD₂ production, whereas HR-EXO exposure increased PTGS2 gene expression. In bEEL, HR-EXO exposure caused a decrease in PGE_2, PGF_2α, PGD₂, PGFM and TXB₂ production. The unique presence of serpin A3-7, coiled-coil domain containing 88A and inhibin/activin β A chain in HR-EXO, indicates potential biomarkers for cows at-risk for metabolic diseases. Our results are in line with the health status of the cow indicating a potential diagnostic role for exosomes in enhancing cows' health and fertility.

Figure 1

Plasma exosomes from transition cows at low-risk (LR-EXO) and high-risk (HR-EXO) for metabolic dysfunction both confirmed exosomal characteristics and differ in exosome particle numbers. (A) Representative western blot for exosomal markers Flotillin 1 (FLOT1) and tumor susceptibility gene 101 (TSG101) of exosomal fractions 5–16 and their presence in exosome-enriched fractions, those exosome-enriched fractions (exosomal samples) were pooled within each animal. (B) Spherical shape was confirmed in electron micrographs of exosomes from exosomal samples. (C) The size of exosomes (nm) is within the defined size of exosome (30–120 nm), and particle numbers (particle per mL) of exosomal samples was higher in low-risk exosomes. Mann-Whitney test was used to identify the significant differences between groups. ***P ≤ 0.001.

Figure 2

Proteomic analysis reveals that protein content differ between plasma exosomes from transition cows at low-risk (LR-EXO) and high-risk (EXO) for metabolic dysfunction. (A) Venn diagram representing unique bovine proteins identifed in LR-EXO (green) or HR-EXO (pink) and common proteins (shaded). Panther analysis revealed differences in bovine proteins from circulating exosomes isolated from LR-EXO cows (B,C) and HR-EXO (D,E) in gene ontology (GO) classifications.

Figure 3

Co-incubation with exosomes from cows at low-risk (LR-EXO) and high–risk (HR-EXO) for metabolic dysfunction lead to changes in eicosanoid expression and secretion in stromal cells. Bovine endometrial stromal cells (bCSC) eicosanoid enzyme gene expressions (A–F) and eicosanoid production (G–L). Values are presented as mean ± SEM. Mann-Whitney test was used to identify the significant differences between groups. *P ≤ 0.05, **P ≤ 0.01.

Figure 4

Co-incubation with exosomes from cows low-risk (LR-EXO) and high–risk (HR-EXO) for metabolic dysfunction lead to changes in eicosanoid expression and secretion in epithelial cells. Bovine endometrial epithelial cells (bEEL) eicosanoid enzyme gene expressions (A–F) and eicosanoid production (G–L). Values are presented as mean ± SEM. Mann-Whitney test was used to identify the significant differences between groups. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.