Differential gene expression in the equine endometrium and conceptus from Day 11 to 16 post-ovulation

Abstract

Mobility of the equine conceptus is essential for maternal recognition of pregnancy (MRP) and is regulated by the conceptus's own derived prostaglandin secretion. Our objective was to compare prostaglandin-related genes and other genes of interest during the expected MRP time. Light breed mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation. Mares in the pregnant (P) group were bred by artificial insemination with >500 million progressively motile normal sperm from one fertile stallion until D0. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Endometrium was collected via a transcervical biopsy punch following conceptus retrieval. Endometrium of non-pregnant (NP) mares was collected at: D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium from the pregnant horn and conceptuses was collected at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). Total RNA from trophoblast and endometrial samples was isolated and evaluated for expression of PTGS2, PTGES, mPGES2, PTGER4, PTGER2, PTGIS, PTGIR, HPGD, SLCO2A1, CRTC2, NR3C1, CBR1, CREB, NF-Kβ, SPP1, IGFBP1, ESR1 and DNTTIP2 using Real-time PCR. Mean threshold cycle was determined and normalized to the reference gene (GAPDH and ACTB) (ΔCT). Statistical analyses were performed using JMP Pro 16, and data was ranked, transformed if necessary, followed by a restricted maximum likelihood model with mare as arandom factor to test differences between Groups and Day. Regarding endometrial mRNA, NP11 had higher expression for NF-Kβ, PTGIS, PTGIR and CBR1 compared to NP13 (p<0.007). ESR1, mPGES2, PTGIS, and CBR1 mRNA were higher in P11 compared to P16 (p<0.05), while CRTC2 was higher in P16 compared to P11 (p<0.06). Lastly, NF-Kβ, PTGIR and CBR1 were higher in P11 compared to P13 (p<0.01), but NF-Kβ P13 was lower compared to P16 (p<0.04). Regarding the conceptuses, CREB and PTGFR expressions were higher in D16 compared to D11 (p<0.003), while CRTC2, NR3C1, NF-Kβ and SPP1 were higher in D16 compared to D11 and 13 (p<0.02). D13 conceptuses had lower expression of HPGD compared to D11 and 16 (p<0.02). Lastly, D11 conceptuses had higher expression of mPGES2, PTGIS and PTGER4 compared to D13 and D16 (p<0.03). Higher expression of D11 PTGIS from both tissues may increase nutrient and blood flow to the endometrium in support of the early conceptus. The upregulation of endometrial NF-Kβ at D11 may be a result of endometrial receptivity through the regulation of inflammation, and the up-regulation of HPGD may function to regulate PGE2 during early pregnancy. Understanding embryonic and endometrial prostaglandin regulation during MRP may help to develop future therapies to ameliorate pregnancy loss in the horse.