Anandamide and Cyclooxigenase-2 Regulate Uterine Stromal Fibroblasts Behavior

CAÑUMIL VA; BELTRAME JS; SORDELLI MS; FRANCHI A; MERESMAN G; RIBEIRO ML

Paris

Congreso; 66th meeting of the Society for Reproductive Investigation,; 2019

Introduction: Anandamide (AEA) is a lipid mediator from the group of endocannabinoids that participates in embryo implantation. AEA tone is regulated by its degrading enzyme, fatty acid amide hydrolase (FAAH) and dysregulations in AEA production are correlated with implantation failure and recurrent abortion. Also, cyclooxigenase-2 derived prostaglandins play a crucial role at implantation sites. During early pregnancy the endometrial stroma is mainly formed by fibroblasts, and later when embryo invasion begins, these fibroblasts develop into decidual cells. Yet, the role of stromal fibroblasts during early gestation remains unexplored. It has been described that anandamide and prostaglandins regulate fibroblast behavior in many biological systems. Using in vitro and in vivo models we investigated the action of anandamide in stromal fibroblasts behavior at the maternal-fetal interface. Methods: As day 5 pregnant rat uterus (day of implantation) is principally composed by stromal fibroblasts, Wistar females were sacrificed in this day of gestation. Uterine horns were excised and incubated for 1 h with AEA 1 nM alone or in the presence of indomethacin (a nonselective cyclooxigenase-1 and 2 inhibitor) or meloxicam (a highly selective cyclooxigenase-2 inhibitor). We determined the production of prostaglandins by RIA. For the in vivo approach, rats in day 5 of gestation received an intra-uterine injection of URB-597 (a FAAH selective inhibitor). Parameters related to implantation events and cyclooxigenase-2 protein level (western blot) were studied in day 8 of pregnancy. In order to validate the results in human stromal fibroblasts, T-hESC cells were seeded in 24 well plates, incubated for 12 h with AEA (1 nM - 1000 nM) or URB-597 (1 nM - 100 nM) and the percentage of wound healing was determined. Results: AEA increased prostaglandins E2 and F2 alpha production (p < 0.05) in day 5 pregnant rat uterus. Pre-incubation with indomethacin or meloxicam reversed AEA stimulation, suggesting that cyclooxigenase-2 mediated anandamide effect. Dysregulated in vivo AEA tone due to treatment with URB-597 increased the percentage of embryonic resorptions (p < 0.05) in 100% of females without modifying the number of implantation sites. Also, in 83% of cases we observed aberrant embryo spacing. Cyclooxigenase-2 protein level was augmented in resorbed implantation sites compared to control. Finally, AEA and URB-597 stimulated T-hESC migration in a concentration-response manner. Conclusion: These results suggest that AEA regulates processes that depend on stromal fibroblasts by a mechanism that involves cyclooxigenase-2. Therefore, deficient AEA-prostaglandins signaling may contribute to endometrial pathologies associated to aberrant stromal fibroblasts migration.