Roles of cytokines and progesterone in the regulation of the nitric oxide generating system in bovine luteal endothelial cells

Abstract

Nitric oxide (NO) produced by luteal endothelial cells (LECs) plays important roles in regulating corpus luteum (CL) function, yet the local mechanism regulating NO generation in bovine CL remains unclear. The purpose of the present study was to elucidate if tumor necrosis factor-α (TNF), interferon γ (IFNG), and/or progesterone (P4) play roles in regulating NO generating system in LECs. Cultured bovine LECs obtained from the CL at the mid-luteal stage (Days 8-12 of the cycle) were treated for 24 hr with TNF (2.9 nM), IFNG (2.5 nM), or P4 (0.032-32 µM). NO production was increased by TNF and IFNG, but decreased by P4 (P < 0.05). TNF and IFNG stimulated the relative steady-state amounts of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein expression (P < 0.05), whereas P4 inhibited relative steady-state amounts of iNOS mRNA and iNOS protein expression (P < 0.05). In contrast, endothelial nitric oxide synthase (eNOS) expression was not affected by any treatment. TNF and IFNG stimulated NOS activity (P < 0.05) and 1400W, a specific inhibitor of iNOS, reduced NO production stimulated by TNF and IFNG in LECs (P < 0.05). Onapristone, a specific P4 receptor antagonist, blocked the inhibitory effect of P4 on NO production in LECs (P < 0.05). The overall findings suggest that TNF and IFNG accelerate luteolysis by increasing NO production via stimulation of iNOS expression and NOS activity in bovine LECs. P4, on the other hand, may act in maintaining CL function by suppressing iNOS expression in bovine LECs.