252 EFFECTS OF LOW CIRCULATING PROGESTERONE CONCENTRATIONS DURING EARLY DIESTRUS ON THE ENDOMETRIAL TRANSCRIPTOME OF THE MARE

Abstract

Progestational-induced changes in endometrial gene expression that are essential for maintenance of pregnancy have been reported in several species. In the horse, serum progesterone (P4) concentrations are routinely measured to assess progestational support of pregnancy and low P4 is implicated as a cause of embryo loss; however, there is little information available concerning changes in the endometrial transcriptome associated with low peripheral P4 concentrations. The objectives of this study were to evaluate changes in the endometrial mRNA transcriptome at Day 12 of the oestrous cycle between mares with normal v. low P4 concentrations. Mares (n = 6) were randomly assigned to control (placebo) or treatment cycles (125 mg of cloprostenol IM on Days 0 through 3 postovulation), and subsequently mares underwent the opposite treatment after a rest cycle. Blood samples were collected daily from the jugular vein. P4 concentrations were measured via ELISA and expressed as area under the curve (AUC). Endometrial biopsies were collected at Day 12 postovulation, RNA was isolated, and the purity and integrity were assessed before RNA sequencing. Library preparation and subsequent nucleotide sequencing were performed according to Illumina protocols (TruSeq Stranded RNAseq Sample Prep kit; Illumina, San Diego, CA, USA). The libraries were quantified by quantitative PCR and sequenced on 2 lanes for 101 cycles from each end of the fragments on a HiSEqn 2000 using a TruSeq SBS sequencing kit version 3, generating an average of 27.3 × 10⁶ stranded 100-bp paired-end reads per sample. The raw sequences (fastq) were analysed using CLC Genomics Workbench software (version 6.0). Trimmed and filtered reads were mapped to the equine reference genome (EquCab2.0) and quantified for nucleotide coordinates corresponding to Ensembl’s consensus gene models (www.ensembl.org). Gene expression analyses were performed using an empirical analysis of digital gene expression and a false discovery rate correction. Gene functions and pathways were analysed using Ingenuity® Pathway Analysis on the differentially expressed transcripts. For the control oestrous cycle, AUC for P4 was 94.7 ± 4.3 (mean ± standard error of the mean) v. 19.6 ± 1.0 ng day^–1 per mL in treated cycles (P < 0.0001). Differential expression of 623 gene loci was identified. Of these, 381 were upregulated and 242 were downregulated in the low-P4 samples. Some of the functions and canonical pathways significantly altered included cholesterol biosynthesis and lipid metabolism and activation of oestrogen mediated S-phase entry, cyclins, and cell cycle: G₁/S checkpoint regulation, oxidative phosphorylation, and apoptosis signaling. In conclusion, lower concentrations of P4 during the luteal phase resulted in substantially altered gene expression and significant changes for several cell-signalling pathways in the equine endometrium.

This work was supported by the Albert G. Clay Endowment and the Mellon Postdoctoral fellowship.