Ovarian steroid-dependent tumor necrosis factor-α production and its action on the equine endometrium in vitro

Tumor necrosis factor-α (TNF) is a cytokine that plays important roles in functions of the endometrium. The aims of this study were to determine whether (i) ovarian steroids modulate TNF production by endometrial cells (Experiment 1); (ii) TNF effects on prostaglandin (PG) production in cultured equine endometrial cells and tissue (Experiment 2). Epithelial and stromal cells were isolated from equine endometrium (Days 2-5 of the estrous cycle; n=20) and treated after passage 1. In Experiment 1, epithelial and stromal cells were exposed to progesterone (P4; 10(-7)M), 17-β estradiol (E2; 10(-9)M) or P4+E2 (10(-7)/10(-9)M) for 24h. Then, TNF mRNA transcription was determined using Real-time PCR. Additionally, TNF protein production was investigated in response to ovarian steroids for 24h using Enzyme-Linked Immunosorbent Spot (EliSpot). In Experiment 2, epithelial and stromal cells and endometrial explants (mid-luteal phase of the estrous cycle; n=5) were exposed in vitro to TNF (10 ng/ml) and to oxytocin (OT; positive control; 10(-7)M) for 24h. The concentrations of PGE2 and PGF2α were determined using a direct enzyme immunoassay (EIA) method. The transcription of prostaglandin-endoperoxide synthase-2 (PTGS-2), prostaglandin E2 synthase (PGES) and PGF2α synthase (PGFS) mRNAs in the endometrial explants was determined using Real-time PCR. Results showed that TNF is produced by two types of equine endometrial cells and its production is up-regulated by ovarian steroids (P<0.05) in stromal cells and by P4 (P<0.05) and E2 (P<0.01) in epithelial cells. Epithelial and stromal cells can also produce PG in response to TNF. In endometrial explants, TNF stimulated PGE2 production to a large extent and PGF2α secretion to a lesser extent. These actions are mediated by up-regulation of PG synthases mRNA transcription. The study indicates that TNF production is closely related to ovarian steroid actions and that the interaction between TNF and PG regulates physiologic processes in the equine endometrium.