PSIII-9 Supplementation of beta-carotene or fatty acids alters production of prostaglandins in bovine endometrial epithelial cells

Differential prostaglandin secretion from the bovine endometrium can be used as a marker for an embryotropic or embryotoxic uterine environment. Beta-carotene has antioxidant properties and is the precursor for retinol, which has been shown to improve early embryonic development in vivo and in vitro. Furthermore, dietary fatty acid supplementation, specifically eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA) has been shown to alter prostaglandin production. The objective of this study was to determine prostaglandin production of endometrial cells following treatment with beta-carotene, EPA, or DHA. Bovine endometrial epithelial cells were treated for 24 hours with serum-free media supplemented with either 10 µM beta-carotene, 10 µM EPA, 10 µM DHA or ethanol (>1% volume/volume) vehicle control. After treatment, concentrations of PGE2 and PGF2a were analyzed in media via commercially available ELISA kits. Concentrations and ratios of prostaglandins were analyzed via ANOVA using the mixed procedure in SAS version 9.4. Beta-carotene treatment decreased PGE2 (P < 0.0001) and PGF2a (P = 0.0003) concentrations in media compared to controls. However, the ratio of PGE2:PGF2a was not different (P = 0.1203) between beta-carotene and controls. DHA treatment decreased PGE2 (P < 0.0001) concentrations in media but did not alter (P = 0.1079) PGF2a concentrations in media compared to controls. The ratio of PGE2:PGF2a was not different (P = 0.6343) between DHA and controls. EPA treatment did not alter (P = 0.1503) PGE2 concentrations in media compared to controls. Conversely, PGF2a concentrations were decreased (P = 0.0088) in media treated with EPA compared to controls. Therefore, the ratio of PGE2:PGF2a was increased (P = 0.0116) between EPA versus controls. These studies demonstrate that in vitro supplementation of EPA may alter the endometrial synthesis of prostaglandins to be more embryotropic. Therefore, EPA may be therapeutic for in vivo trials to influence the early uterine environment.

Functionally Selective Inhibition of the Oxytocin Receptor by Retosiban in Human Myometrial Smooth Muscle

Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared with first-generation oxytocin antagonists when used for the prevention of preterm birth. The aim was to determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban compared with the currently used peptide-based compound atosiban. Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Retosiban antagonism of oxytocin action in human myometrium was potent, rapid, and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single-site competitive binding kinetics for epelsiban, retosiban, and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban but not retosiban inhibited forskolin, and calcitonin stimulated 3′,5′-cyclic adenosine 5′-mono-phosphate (cAMP) production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated extracellular regulated kinase (ERK)1/2 activity in a time- and concentration-dependent manner. Oxytocin and atosiban stimulated cyclo-oxygenase 2 activity and subsequent production of prostaglandin E2 and F2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gα q G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gα i G-proteins. Retosiban and epelsiban demonstrate distinct pharmacology when compared with atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production.