The effect of tumor necrosis factor alpha (TNFα) on in vitro steroidogenesis in two strains of White Leghorn hens

Two strains of White Leghom hens selected for high (HA) or low (LA) antibody response to challenge with sheep red blood cells were used to determine differences in circulating levels of progesterone (P 4) and luteinizing hormone (LH), and to determine in vitro effects of tumor necrosis factor alpha (TNF a) on granulosa cell steroid secretion during different periods of the estrous cycle. In experiment one, hens were serially bled from 4 hours after oviposition until the next oviposition. Plasma was analyzed for P 4 and LH using validated radioimmunoassays (RIA's). In the second experiment, hens were sacrificed at 16 and 37 weeks of egg production. Granulosa cells isolated from the F1, F2, and F3 follicles were incubated for 3 hours in the presence of; 1) untreated RPMI 1640 + 0.1% bovine serum albumin + 1% antibiotics (control) 2) 5 ng/ml human recombinant TNFa (hTNFa) 3) 5 ng/ml murine recombinant TNFa (mTNFa) 4) 100 ng/ml ovine LH (oLH) 5) hTNFa plus oLH 6) mTNFa plus oLH. Conditioned media was analyzed for P 4 and P 5 secretion as before. Plasma levels of both P 4 and LH were significantly higher (P~0.05) among HA hens during the peak preovulatory period of the estrous cycle (-6 to -1 hours before oviposition). Circulating levels of P4 were 1.45, 2.19, 2.67, 2.66, and 2.38 ng/ml in LA hens versus 1.82, 3.05, 3.19, 3.07, 3.01, and 2.79 ng/ml respectively in HA hens at 6, 5, 4, 3, 2, and 1 hour prior to oviposition. Levels of LH were 2.36, 3.36, 3.57, 2.81, 2.41, and 1.95 ng/ml in LA hens versus 3.58, 5.27, 5.38, 4.51, 3.77, and 3.71 ng/ml respectively in HA hens for the same period. As with plasma, granulosa cells from HA hens produced more (P~0.05) P4 and P5 than did those from LA hens. Similarly, granulosa cells from larger follicles secreted more (P~0.05) P4 and P5 than did those from smaller follicles. Treatment of granulosa cells with human, but not murine, TNFa reduced (P~0.05) basal P 4 and P5 production by granulosa cells from preovulatory follicles. Conversely, treatment of cells with oLH significantly increased (P~0.05) P4 and P5 as compared to controls. The combination of mTNFa., but not h1NFa., and oLH consistently stimulated (P~0.05) P4 and P5 secretion above that observed with treatment of oLH alone. Mouse 1NF a. is known to activate both type I and type II 1NFa. receptors, while h1NFa. binds only the type I receptor. This may explain the differential effects on in vitro steroid secretion observed through use of the two forms of 1NFa. in these experiments. Furthermore, the observation that h1NFa. and oLH rarely caused significant increases, but frequently caused numerical increases in P 4 and P 5 secretion is suggestive of a ligand passing role for the type II receptor. No clear pattern emerged as to when during the estrous cycle h1NFa. affected steroid production. In the avian, reduced steroidogenic capacity (specifically, the production of progesterone) can lead to delayed or inhibited ovulation. Results from these experiments suggest a potential role for human and/or murine 1NFa. in reproduction in the laying hen (Gallus domesticus).