62 Peripheral transcriptome response to embryo mortality in Holstein cows

Abstract

Exactly how failure through conceptus-endometrial signalling on Day 16 of pregnancy causes early embryo mortality (EM) is yet unknown. It was hypothesised that EM pregnancies are associated with an impaired endocrine action of interferon tau (IFNT) from the conceptus on the corpus luteum (CL) and peripheral blood mononuclear cells (PBMC), in addition to paracrine failure in preventing the upregulation of oestradiol-mediated luteolytic signals in the endometrium. Cows not exposed to semen served as controls on Day 16 of the oestrous cycle (EC; n=7). Conceptuses were flushed on Day 16 of pregnancy and were then sorted based on quality. Normal (N, n=9) conceptuses were elongated and translucent, whereas EM conceptuses (n=6) lacked extensive elongation and were pink, red, or opaque. The RNA extracted from conceptuses, endometrium, CL, and PBMC was submitted to RNA-Seq analysis. Trimmed and aligned sequences were analysed in R using the DESEqn 2 package. Data were submitted to ingenuity pathway analysis with fold change ≥1.5 and P ≤ 0.05. Previously, we reported that N were longer (P<0.006) than EM conceptuses. The IFNT mRNA transcript raw counts (from RNA-Seq) were greater (P<0.0001) in N than in EM conceptuses. The IFNT protein concentrations (enzyme-linked immunosorbent assay) in uterine flushings were greater (P<0.004) in N compared with EC and tended (P<0.07) to be greater in EM compared with EC cows. Similarly, interferon stimulated gene 15 (ISG15) mRNA raw counts were greater (P<0.003) in N and tended (P<0.09) to be greater in EM endometrium compared with EC endometrium. Ingenuity pathway analysis revealed that T helper (Th1) cell activation, a key canonical pro-inflammatory pathway, was upregulated in EM compared with N conceptuses. Among the 15 upregulated endometrial genes in EM compared with N, 9 were associated with oestradiol action. In the current study, 7/9 PBMC (ISG15, MX2, OAS1, OAS2, IFI44, IFI6, and DDX58) and 3/3 CL (ZBP1, OAS1, and MX1) genes (ISGs) were upregulated in N versus EC and belonged to the interferon signalling canonical pathway. Key upstream regulators were IFNAR2 and IRF7 for PBMC and type I IFNs and ISG15 for CL. There were no differences in EM compared with N genes in PBMC. Conversely, 10 genes in the CL were upregulated for EM compared with N and aligned to pro-inflammatory/calcium binding (i.e. CAPS2, S100A12) and inhibition of adenylate cyclase (HTR1B) responses. The primary canonical pathways were granulocyte adhesion and diapedesis, and hypercytokinemia. Moroever, key upstream regulators were MAPK and TNF. In conclusion, ISGs are upregulated peripherally in PBMC and CL in response to normal pregnancy. When conceptuses are compromised, pro-inflammatory and calcium-mediated responses that promote a localised “cytokine storm” immune response are upregulated and may be coupled with disruption of steroidogenesis in the CL, which is mediated by adenylate cyclase. This research was supported by USDA-Agriculture and Food Research Initiative (AFRI)-National Needs Graduate and Postgraduate Fellowship (NNF) 2016-38420-25289 and the W3112 USDA-AFRI Ruminant Reproduction Project.