12 Impact of progesterone signaling on tumor necrosis factor-alpha synthesis by luteal macrophages

Abstract

Abstract Understanding the mechanism in which luteolysis occurs is an important step in improving the reproduction of livestock in the industry. The corpus luteum (CL) is a transient endocrine organ found on the ovary responsible for progesterone secretion. It is well known that prostaglandin F2-alpha (PGF) is responsible for luteal regression, however, the process by which luteal cells (LC) become sensitive to the luteolytic effects of PGF remain unclear. Therefore, the objective of this study was to determine if suppression of progesterone (P4) synthesis or blocking P4 signaling activates tumor necrosis factor (TNF)-alpha production by the luteal macrophages. Ovaries were collected from an abattoir and transported to the laboratory on ice. Ovary status (i.e. approximate days post ovulation), was determined by phenotypic description of ovarian structures. Only ovaries (n = 5) containing midcycle (d 12/13 post ovulation) CL were used in these experiments. Luteal tissue was isolated and dissociated to collect LC and and luteal macrophages (MAC). Isolated cells were incubated independently at 37°C at 5% CO2 for 24 h in 24-well plates. Luteal cells and MACs were then harvested and placed in the following conditions: 1) LC + MAC + DMSO (control), 2) LC + MAC + trilostane (TRIL 1.0mg/mL), 3) LC + MAC + TRIL 1.5 mg/mL, 4) MACS only, 5) MACS +LPS. Supernatant was harvested at 24 h whereas both cells and supernatant were harvested after 48 h, in vitro. Supernatant was immediately frozen for subsequent P4 analysis, via RIA. Data results were analyzed using a MIXED procedure of SAS and evaluated the effects of cell-type, treatment, and culture time. Statistical significance was determined at P < 0.05 and a tendency at 0.05 < P < 0.10. Overall, TRIL treatment reduced supernatant P4 concentration (8.3 and 6.8 ng/mL to 1.0 and 1.5 mg/mL TRIL, respectively) compared with controls (22.8 ng/mL; P = 0.0012); however there was not a difference when comparing the TRIL treated cultures. Progesterone concentrations tended to decrease over time (P = 0.0661). At 24 h in vitro, P4 concentrations were suppressed in 1.0 mg/mL TRIL and 1.5 mg/mL TRIL treated wells (5.3 and 4.2 ng/mL, respectively), compared with controls (16.4 ng/mL; P = 0.0014). Whereas P4 concentrations were reduced in the 1.5 mg/mL TRIL treated wells (1.6 ng/mL) compared with controls (9.95 ng/mL; P = 0.0419) and tended to differ in the1.0 mg/mL TRIL treated wells (2.4 ng/mL; P = 0.0634), at 48 h in vitro. Progesterone concentrations were undetectable in wells containing MAC only. Collectively, TRIL treatment effectively reduced P4 synthesis in LC cultures. Therefore, the greater concentration of TRIL is needed for P4 suppression in longer term culture systems. Additional analyses will be conducted to determine the effect of P4 suppression on macrophage function.