Global endometrial transcriptomic profiling: transient immune activation precedes tissue proliferation and repair in healthy beef cows

Abstract

Background: All cows experience bacterial contamination and tissue injury in the uterus postpartum, instigating a local inflammatory immune response. However mechanisms that control inflammation and achieve a physiologically functioning endometrium, while avoiding disease in the postpartum cow are not succinctly defined. This study aimed to identify novel candidate genes indicative of inflammation resolution during involution in healthy beef cows. Previous histological analysis of the endometrium revealed elevated inflammation 15 days postpartum (DPP) which was significantly decreased by 30 DPP. The current study generated a genome-wide transcriptomic profile of endometrial biopsies from these cows at both time points using mRNA-Seq. The pathway analysis tool GoSeq identified KEGG pathways enriched by significantly differentially expressed genes at both time points. Novel candidate genes associated with inflammatory resolution were subsequently validated in additional postpartum animals using quantitative real-time PCR (qRT-PCR).

Results: mRNA-Seq revealed 1,107 significantly differentially expressed genes, 73 of which were increased 15 DPP and 1,034 were increased 30 DPP. Early postpartum, enriched immune pathways (adjusted P < 0.1) included the T cell receptor signalling pathway, graft-versus-host disease and cytokine-cytokine receptor interaction pathways. However 30 DPP, where the majority of genes were differentially expressed, the enrichment (adjusted P < 0.1) of tissue repair and proliferative activity pathways was observed. Nineteen candidate genes selected from mRNA-Seq results, were independently assessed by qRT-PCR in additional postpartum cows (5 animals) at both time points. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes were significantly elevated 30 DPP and are functionally associated with tissue repair and the restoration of uterine homeostasis postpartum.

Conclusions: The results of this study reveal an early activation of the immune response which undergoes a temporal functional change toward tissue proliferation and regeneration during endometrial involution in healthy postpartum cows. These molecular changes mirror the activation and resolution of endometrial inflammation during involution previously classified by the degree of neutrophil infiltration. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes may become potential markers for resolution of endometrial inflammation in the postpartum cow.

Figure 1

Work flow for next generation sequencing data analysis, the resulting gene expression output from TMM EdgeR and downstream analysis with differentially expressed genes. Sequenced reads were mapped to the bovine genome with TopHat, summarised with HtSeq Count and normalised with TMM EdgeR. Output files of differentially expressed (DE) genes with P < 0.05 (adjusted P < 0.45) and an adjusted P < 0.1 were used in GoSeq pathway analysis. Nineteen of the most highly significant DE genes, (adjusted P < 0.1) 15 days and 30 days postpartum, were selected for assessment by qRT-PCR in a larger sample set (n = 5).

Figure 2

Differentially expressed genes within the Wnt Signalling pathway. Genes significantly elevated (P < 0.05) 15 DPP are in green and 30 DPP are in red.

Figure 3

Differentially expressed genes within the ECM (extra cellular matrix) receptor interaction pathway. Genes significantly elevated (P < 0.05) 15 DPP are in green and 30 DPP are in red.

Figure 4

Log₂ fold changes from quantitative real-time PCR and mRNA-Seq for 19 differentially expressed genes in the endometrium of healthy cows between 15 and 30 days postpartum (DPP). Genes selected were significantly differentially expressed by mRNA-Seq (dark grey bars) (adjusted P < 0.1) and the P-values on the graph are those for qRT-PCR results (light grey bars) (* = P < 0.05, ** = P < 0.005). The error bars are representative of the standard error of the mean (SEM). Genes denoted by # have 0 reads at one time point across all samples and for illustrative purposes are represented on this graph as twice the size of the largest Log₂ fold change. Log₂ fold changes to the left of 0 are increased 15 DPP and Log₂ fold changes to the right of 0 are increased 30 DPP.