Effect of variants of interferon-τ with mutations near the carboxyl terminus on luteal life span in sheep

Abstract

Mutant forms of recombinant ovine interferon-τ (oIFN-τ) have been previously prepared by site-directed mutagenesis of an ovine gene with the purpose of establishing relationships between structure and function of the molecule. These mutant forms have altered antiviral and antiproliferative activities, and receptor-binding affinities, and their ability to extend estrous cycle length after being injected i.m. into nonpregnant ewes has been established. In experiment 1, i.m. injection of either PBS (vehicle) alone or with 0.1 mg (n = 4), 0.5 mg (n = 4), or 2.0 mg (n = 3) of recombinant oIFN-τ (S4, identified below) was performed twice daily on Days 11-18 postestrus (Day 0 = estrus). Luteal life span was extended (p < 0.05) by 4.8 days after injection of 0.5 mg or 2.0 mg oIFN-τ; injection of 0.1 mg had no effect (p > 0.05) on luteal life span relative to the group that received vehicle alone. In subsequent experiments, the dosage of oIFN-τ was 1.0 mg per injection twice daily. The objective of experiment 2 was to determine the effect of mutated forms of oIFN-τ on luteal life span in sheep. Fifty-seven ewes received twice-daily injections (i.m.) from Day 10 to Day 20 postestrus of PBS (vehicle) either alone (n = 9), with 0.3 mg/injection of bacterial contaminating proteins (BP; n = 10), or with 1 mg/injection of one of the following forms of recombinant oIFN-τ 1) a fully active, 172-amino acid (aa) oIFN-τ (S4, n = 10); 2) a form lacking 11 aa at the carboxyl terminus and with an [143→T mutation (S1), which had very low antiviral and antiproliferative activities and receptor-binding affinities (n = 9); 3) a truncated form identical to S1 but with I143 restored (TRN11), which had low antiviral and antiproliferative activities but only slightly reduced receptor-binding affinities (n = 10); and 4) a form similar to wild type S4 (S4-K) with low antiviral and antiproliferative activities but only slightly reduced receptor-binding affinities (n = 9). Luteal life span was slightly longer (p < 0.05) in the TRN11 and S4-K groups (19.6 and 20.6 days, respectively) than in the PBS, BP, and S1 groups (16.2, 16.8, and 17.5 days, respectively). In the S4 group, mean luteal life span was 33.6 ± 5.9 days (range 15.5-64 days). A third experiment entailing twice-daily injections of either 0.3 mg BP (n = 6), 1 mg TRN11 (n = 5), 1 mg S4-K (n = 5), or 1 mg S4 (n = 5) was conducted in which the pyrogenic effect of the oIFN-τ was also examined. Luteal life span was longer (p < 0.05) in the S4-K and S4 groups (18.9 and 28 days, respectively) than in the BP and TRN11 groups (16.6 and 17.6 days, respectively). Intramuscular injection of all forms of IFN-τ caused hyperthermia in ewes initially, but ewes appeared to become refractory to treatment after several days. In summary, extension of luteal life span was more closely associated with biological activity as assessed in vitro than with receptor binding affinities.