Effects of Oxytocin, LPS, and Polyunsaturated Fatty Acids on PGF2α Secretion and Gene Expression during Equine Endometrial Culture

s / Journal of Equine Veterinary Science 31 (2011) 230-356 314 Reproductive Physiology: Graduate Student Competition Effects of Oxytocin, LPS, and Polyunsaturated Fatty Acids on PGF2a Secretion and Gene Expression during Equine Endometrial Culture L.V. Penrod , R.E. Allen , M.L. Rhoads , J.L. Turner , S.W. Limesand , and M.J. Arns 1 1 The University of Arizona, Tucson, AZ, USA, New Mexico State University, Las Cruces, NM, USA Introduction: Early embryonic loss in the mare can be caused by a lack of maternal inhibition of prostaglandin F2a (PGF2a) or increased secretion of PGF2a due to uterine inflammation. Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been shown to decrease PGF2a release [1]. In this study, our objectives were to determine arachidonic acid (AA), EPA, and DHA uptake in equine endometrial explants and to evaluate their influence on PGF2a secretion and expression of enzymes involved in prostaglandin synthesis. Materials and Methods: In experiment one, two endometrial biopsies from 4 mares were collected on d 2 of estrus. Biopsies were minced and equally divided into 5 wells (35 mm) and incubated in Hams F-12/MEM media for 90 min at 37 C in a humidified atmosphere with 5% CO2. Rinsed explants were placed in one of the following treatments: control (serum free media) or 100 mM AA, EPA or DHA. After 24 h of culture, explants were stored at 80 C until analysis of fatty acid (FA) uptake was performed using gas liquid chromatography. For experiment two, four endometrial biopsies taken from8mares were divided into 24 sections and plated into individual wells on multiple plates. Each of four wells on a plate received one of the following treatments in 2.5 mL of serum free media: control (no treatment),100 mMAA,100 mMEPA, or 100mMDHA. Explantswere then incubated for 24 h to allow for FA uptake. Following incubation, 2 plates served as control and received no stimulant, 2 plates were stimulated with oxytocin (250 nM), and 2 plates were stimulated with lipopolysaccaride (LPS, 1 mg/mL). Media was collected at 6 h and stored at 80 C until PGF2a analysis using EIA kits (Cayman Chemical Co., AnnArbor,MI). The relative expression of cyclooxygenase-1 and 2 (Cox-1 and 2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and phospholipase A2 (PLA2)mRNA transcripts weremeasured by quantitative real-time PCR. Threshold cycles were normalized to a reference gene (ribosomal protein S15) using the comparative D Ct method and fold changes (DD Ct) were determined as previously described [2]. The effects of treatment, time, stimulant and their interactions on PGF2a concentrations were analyzed using the PROC MIXED procedure of SAS [3]. Mare was considered random for all experiments. Results and Discussion: Prostaglandin F2a concentrations were higher (P < .0001) in explants challenged with oxytocin or LPS as compared to controls despite FA addition (Figure 1). Moreover, oxytocin stimulated explants to a greater (P< .007) extent than LPS. Endometrial explants incorporated EPA (P < .05) and DHA (P < .01) and tended (P < .06) to uptake AA. When explants were stimulated with oxytocin or LPS, DHA inhibited (P < .0001) PGF2a secretion, whereas AA and EPA failed to influence PGF2a secretion. Oxytocin increased (P < .02) expression of Cox-1, Cox-2, PGES, and PLA2 as compared to controls regardless of FA treatment. Similarly, LPS increased (P< .03) expression of Cox-2, PGFS, PGES, and PLA2. Discussion: Herein, we report oxytocin stimulated PGF2a release from endometrial explants is due to an up-regulation of Cox-1 and 2, PGES, and PLA2 expression. The up-regulation of Cox-2 is similar to reports in the porcine [4] and equine [5]. For the first time we have shown LPS stimulates PGF2a release from explants through up-regulation of Cox-2, PGFS, PGES, and PLA2. SupplementationwithDHAorEPAhasbeenshowntodecreasePGF2a secretion [1],whereasAAhasbeenshownto increasereleaseofPGF2a [1,6]. In this study, DHA inhibited PGF2a release, although EPA andAA had no influence. Inhibition of PGF2a by DHA was not due to down regulation of enzyme expressionwhich is similar to that reported for bovine endometrial tissue [1]. Differences in these studies may be attributed to cell type and/or culture conditions. In conclusion, upregulation of gene expression is onemechanism for oxytocin and LPS stimulated PGF2a release. Inhibition by DHA is through another mechanism other than down regulation of gene expression.