Expression pattern of inflammatory response genes and their regulatory micrornas in bovine oviductal cells in response to lipopolysaccharide: implication for early embryonic development.

Ernst Tholen,Dawit Tesfaye,Michael Hoelker,Shuhong Zhao,Sally Ibrahim,Franca Rings,Christiane Neuhoff,Christian Looft,Dessie Salilew-Wondim,Karl Schellander

doi:10.1371/journal.pone.0119388

Abstract

In the present study, we used an in vitro model to investigate the response of the oviduct with respect to inflammatory mediators and their regulatory microRNAs in case of bacterial infection and subsequent association with embryo survival. For this, we conducted two experiments. In the first experiment, cultured primary bovine oviductal cells (BOEC) were challenged with lipopolysaccharide (LPS) for 24h and the temporal expression pattern of inflammatory mediators and their regulatory microRNAs were measured at 0, 3, 6, 12, 24 and 48h after LPS treatment. Intriguingly, the temporal patterns of all miRNAs except miR-21 were significantly up-regulated at 6h after LPS treatment. Whereas, we observed significant overexpression of pro-inflammatory mediators as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) after LPS challenge for 24h. On the other hand, the expression level of essential elements like oviductal glycoprotein 1 (OVGP1) and insulin-like growth factor 2 (IGF2) was significantly decreased in challenged groups compared with control. Moreover, miR-155, miR-146a, miR-223, miR-21, miR-16 and miR-215 have shown a clear suppression in challenged group after LPS treatment. In the 2nd experiment there were four groups of blastocysts produced, namely embryo+LPS free media, embryo+LPS, BOEC+embryo and BOEC+embryo+LPS. The suboptimal oviduct environment due to LPS challenge is found to have a significant influence on the expression of inflammatory response genes (TNFα and CSF1), stress response genes (SOD and CAT), mitochondrial activity, reactive oxygen species (ROS) accumulation and apoptotic level either in cultured or co-cultured blastocysts. Collectively, LPS challenge led to aberrant changes in oviductal transcriptome profile, which could lead to a suboptimal environment for embryo development.

Highlights

In cattle, bacterial contamination of the uterine lumen is ubiquitous after parturition, and up to 40% of animals develop pelvic inflammatory disease (PID) and 20% have endometritis [1,2]
The relative abundance of IL1β, TNF receptor associated factor 6 (TRAF6), TNFα, CASP3, TGFβ1 and superoxide dismutase 1 (SOD) was increased significantly (0.001 p 0.01) and the expression level of oviductal glycoprotein 1 (OVGP1) and insulin-like growth factor 2 (IGF2) was decreased significantly in challenged group compared with control (Fig. 2)
The expression level of some miRNAs was found to show a reciprocal pattern to their target genes (TRAF6, IL1β, TGFβ1 and TNFα), whereas the expression level of some miRNAs were found to have a similar pattern to their target genes (IGF2, OVGP1 and INOS), (S2‒S4 Figs.)

Summary

Introduction

Bacterial contamination of the uterine lumen is ubiquitous after parturition, and up to 40% of animals develop pelvic inflammatory disease (PID) and 20% have endometritis [1,2]. Oviduct is the female genital organ at which oocyte maturation, sperm capacitation, fertilization and transport of gametes and embryos is occurring (maturation, capacitation etc do not occur in other organ), [6] and the disturbance of oviduct function due to pathogenic infection could result early embryonic loses and infertility. Despite the mucosal role in immune defence of the genital tract, little is known about the mechanism of bovine oviduct epithelial cell activation by pathogens or about the receptors and secondary mediators involved in this response. It is well established that Toll-like receptors (TLRs) have important roles in detecting pathogens and in initiating inflammatory responses that subsequently prime specific adaptive immune responses during infection [11]. Binding of TLR4 to LPS initiates a cascade of signaling events that evokes the production of cytokines and pro-inflammatory mediators [13]. The functional role of TLR4 in the intact endometrium and oviduct of human, mouse and in the intact endometrium of bovine has been explored, less is known about its role in bovine oviduct [10,14,15]

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Conclusion