PS4-17. Evaluating Expression Patterns of Uterine ISG15 Post-embryo Transfer in Beef Heifers.

Abstract Early pregnancy detection in cattle is crucial to reduce the time between calving and conception and improve farm profitability. Interferon stimulated genes (ISGs) can be used as an early diagnostic marker for pregnancy establishment. Previous work has reported the expression of interferon stimulated gene 15kDa (ISG15) within the bovine uterine endometrium. However, expression of ISG15 between regions of the uterine endometrium have not been fully analyzed in cattle. Therefore, the objective of this study was to compare expression patterns of ISG15 between caruncular and intercaruncular endometrial tissues post-embryo transfer in beef heifers. Crossbred beef heifers (n = 18) received in vitro produced embryos on day 8 post-estrus following a 7-day Co-Synch estrus synchronization protocol. Blood was collected via coccygeal venipuncture on day 26 and centrifuged at 2,500 x g and 4 °C for 15 minutes. Resulting plasma was collected and stored at -20 °C for progesterone (P4) analysis. Heifers were then necropsied and ovariohysterectomized. Caruncular (C) and intercaruncular (IC) endometrial tissues were collected from the ipsilateral uterine horn, individually snap-frozen in liquid nitrogen, and stored at -80 °C for subsequent RTqPCR with the target gene of ISG15. Cross-sections of both the ipsilateral and contralateral (relative to the corpus luteum) uterine horns were collected and fixed in 4% paraformaldehyde prior to paraffin embedding and sectioning. Immunofluorescence analyses were performed to colocalize ISG15 with epithelial-cadherin (E-Cad). Relative gene expression for each tissue was analyzed using one-way ANOVA and Tukey's HSD for pairwise comparisons, respectively, in JMP Pro 19.0. Data correlations of mean relative mRNA expression of ISG15 by circulating concentrations of P4 were analyzed using Spearman's correlations in PROC CORR in SAS 9.4. There was no difference in mean relative mRNA expression of ISG15 between C (1.43 ± 0.43) and IC (1.98 ± 0.43; P = 0.37). Additionally, there was no significant correlation between mean relative mRNA expression of ISG15 within both C and IC and circulating concentrations of P4 on day 26 (r=-0.22; P = 0.24). There were no conceptuses present upon necropsy. However, immunofluorescence analyses localized ISG15 within the apical perinuclear space of uterine glandular epithelium in both the stratum spongiosum and stratum compactum of the ipsilateral horn. Additionally, ISG15 localized within the tunica media of blood vessels in the ipsilateral horn. There was no immunostaining of ISG15 within the contralateral uterine horns. These results suggest that the expression of ISG15 within C and IC tissues may be indicative of an immune response given the absence of a conceptus upon necropsy. Alternatively, residual influence from the transferred embryo may have elicited the expression of ISG15 within the ipsilateral glandular epithelium. Further analysis of uterine ISG15 expression following embryo transfer should be evaluated to determine if expression of ISG15 is conceptus driven or an unrelated immune response.