Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy

Abstract

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2α (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14–16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO (n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 (P ≤ 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13.

Maternal Recognition of Pregnancy in the Horse: Are MicroRNAs the Secret Messengers?

“…Transcriptomics of the equine endometrium showed differential expression of 245 genes. A similar approach has been published previously in the horse, using microarray or RNA-Sequencing to evaluate pregnancy associated endometrial gene expression between day 8 and day 18 [19,21,22,24,36]. Yet, another transcriptomics by RNA-Sequencing in our study provided added value as it allowed to build the bridge between proteomics and miRNA expression on day 13 and as RNA-Sequencing is more sensitive than microarray.…”

“…Differential expression between pregnant and cyclic mares was found for 12 miRNAs in the endometrium and for two miRNAs in the UF. Research on the role of miRNAs during equine pregnancy is limited to a few studies focused on later stages of gestation [32,33], a recent study assessing non-coding RNAs in the endometrium in which no differences were found on day 13 [35] and one study determining differential expression of miRNAs in serum of pregnant vs. cyclic mares on day 9, day 11, and day 13 [34]. Two common miRNAs were detected.…”

“…High throughput molecular biology at the embryo-maternal interface has substantially contributed to the knowledge on pathways affected during MRP. Technological advantages, including sequencing, favored development of transcriptomics and several studies assessed gene expression of the endometrium and the conceptus between days 8 and 16 of pregnancy [19][20][21][22][23][24]. While no differences between pregnant and cyclic mares were detected on day 8, analysis on days 12 and 14 indicated pregnancy related upregulation of genes involved in nutrient supply and endometrial receptivity and downregulation of trophoblast invasion factors [14]; by day 16, clear differences were established.…”

“…A potential signaling role during MRP was assessed first by quantitative PCR to examine differential expression of miRNAs in serum; this indicated pregnancy related expression of miRNAs targeting focal adhesion molecules and expression of selected target mRNAs and proteins was evaluated in the endometrium [34]. Recently, the same group assessed expression of non-coding RNAs in the equine endometrium, and even though 419 differentially expressed non-coding RNAs were found on day 11, no differential expression was detected on day 13 [35].While all disciplines of molecular biology have contributed to the current knowledge on MRP in the horse, a holistic approach in which high-throughput proteomics, transcriptomics and miRNA expression at the embryo-maternal interface can be linked directly is currently lacking. The aim of this study was to provide such integrated analysis.…”

“…A potential signaling role during MRP was assessed first by quantitative PCR to examine differential expression of miRNAs in serum; this indicated pregnancy related expression of miRNAs targeting focal adhesion molecules and expression of selected target mRNAs and proteins was evaluated in the endometrium [34]. Recently, the same group assessed expression of non-coding RNAs in the equine endometrium, and even though 419 differentially expressed non-coding RNAs were found on day 11, no differential expression was detected on day 13 [35].…”