(164) Localization of Immune Markers at the Bovine Uteroplacental Interface During Early Gestation

Dallas R. Soffa¹; Joe W. Cain^2,3; Heewon Seo³; Michael F. Smith⁴; Julie A. Walker⁵; Thomas W. Geary⁶; George A. Perry⁷; Gregory A. Johnson²; Rebecca K. Poole¹

1. Department of Animal Science, Texas A&M University, College Station, USA


2. Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, USA


3. Department of Animal and Avian Sciences, University of Maryland, College Park, USA


4. Department of Animal Sciences, University of Missouri, Columbia, USA


5. Department of Animal Science, South Dakota State University, Brookings, USA


6. Fort Keogh Livestock and Range Research Laboratory, USDA-ARS, Miles City, USA


7. Texas A&M AgriLife Research and Extension Center, Texas A&M University, Overton, USA

The complex microanatomical changes paired with dynamic immune/inflammatory profiles at the uteroplacental interface during early pregnancy may contribute to gestational failure in cattle. Previous research has shown that interferon-stimulated genes (ISGs) within the uterus are upregulated during early pregnancy in response to interferons produced and secreted by the conceptus trophectoderm. As a response to the semi-allogeneic conceptus, such ISG upregulation within the uterine tissues may influence the immunological status of the intrauterine environment during implantation. However, the specific localization of immune markers at sites of implantation throughout early pregnancy in cattle has yet to be elucidated.  Therefore, this study aimed to detect and localize ISG15 and toll-like receptor (TLR) 4 at the bovine uteroplacental interface during early gestation in beef cattle. Crossbred beef cows (n=62) were synchronized utilizing the 7-day CO-Synch + CIDR protocol. All cows were artificially inseminated on d0 with semen from a single sire. Cows were harvested and ovariohysterectomized on d20, d26, and d32. Cross-sections of the uterine horn, ipsilateral to the corpus luteum, of suspected pregnant tracts (as determined by ultrasonography) 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), and TLR4 with E-Cad. Interferon-stimulated gene 15 and TLR4 were detected across all days of pregnancy examined. On d20, ISG15 was localized to endothelial cells within the stratum compactum stroma of both caruncular and intercaruncular regions. Expression of ISG15 was also detected within the endothelial cells surrounding endometrial glands deeper in the stratum spongiosum. Similar expression of ISG15 was detected on d26, with distinct localization in stromal areas beneath the uterine luminal epithelium (LE) more proximal to the developing conceptus. Localization of ISG15 appeared to shift from the stratum compactum stroma to the glandular epithelium (GE) on d32 of gestation. Toll-like receptor 4 was localized to the basal region of the uterine LE, throughout the cytoplasm of the GE, and interspersed throughout the developing trophoblast on d20.  Expression was also detected in endothelial cells and the tunica media of larger blood vessels. On d26 of gestation, TLR4 was localized throughout the cytoplasm of trophoblast cells and within the GE. On d32, TLR4 was still detected in the GE and the trophoblast cells, but also appeared throughout areas of the extra embryonic endoderm. Epithelial-cadherin was localized to the uterine LE and the binucleate giant cells of the trophoblast throughout all days of gestation studied. However, scattered localization of E-Cad throughout the uterine LE suggests that placentation may cause remodeling not typically associated with the epitheliochorial placenta type in cattle. Collectively, this research is the first to identify the presence and localization of ISG15 and TLR4 at the uteroplacental interface during early gestation in cattle. These results enhance our understanding of the intricacies of the establishment and maintenance of early stages of gestation, providing insights into potential physiological strategies to improve reproductive efficiency in cattle. This research was funded by the 2024 SSR Emerging Investigator Grant titled “Unraveling the influence of estradiol on reproductive tract microbiota and fertility in beef cattle” and NIFA 2019-67015-29411.