Characterization of interferons in the equine conceptus and endometrium

Abstract

Interferons (IFN) are involved in the maintenance and establishment of pregnancy in a variety of species. There are three types of IFNs, which include type I (IFN-α, β, ε, τ, and δ), type II (IFN-γ), and type III (IFN-λ1, λ2 and λ3). In the horse, IFNA, IFND and IFNO are expressed in the equine trophoblast, and IFNE is expressed in higher abundance in equine endometrium during the luteal phase. However, investigation of IFNs and their receptors during early equine pregnancy is still lacking. Our objective was to investigate the expression of IFN types I and II and their receptors, and other IFN related genes in equine conceptuses and endometrium during early pregnancy. Mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation (OV). Pregnant (P) group mares were bred using artificial insemination by a single fertile stallion. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Following conceptus retrieval, an endometrial biopsy was collected. Endometrial samples from nonpregnant (NP) mares were collected at D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium and conceptuses were collected from pregnant mares at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). As the majority of nonpregnantmares would have undergone luteolysis, a D16 sample was not included in this study. Total RNA was isolated and evaluated for expression of interferon-related genes using Real-time PCR. The mean threshold cycle was determined and then normalized to the mean of the reference genes (GAPDH and ACTB) (ΔCT). A restricted maximum likelihood model was performed in JMP Pro 16 (Cary, NC) with mare as a random factor with significance set at P<0.05. There was no amplification for IFNB and IFND in the endometrium, and no amplification of IFNG, IFNB and IFNAR2 in the conceptus. In the endometrium, IFNAR1 and IFNGR1 had higher expression in P11 compared to P13 (P<0.02). Expression of endometrial IFNG mRNA was higher in P11 and P16 compared to P13 (P<0.004). There was no difference in the mRNA relative abundance of endometrial ISG15 and IFITM1 between groups or days. In the conceptus, IFNA, IFNO, and IFNGR2 expression were higher in D16 compared to other ages (P<0.05). D11 and D16 conceptuses had higher expression of IFNAR1 compared to D13 (P<0.0002), while IFNGR1 was higher in D16 compared to D11 (P<0.004). The role of interferons during maternal recognition of pregnancy in the horse is unknown. Unlike the pig, the horse embryo does not seem to express IFNG. Further investigation is underway.