57 NO BENEFICIAL EFFECT OF AGGREGATION ON INTERFERON TAU ACTIVITY IN BOVINE CLONE OUTGROWTHS

Abstract

Extraembryonic failure is associated with the early developmental failure of bovine somatic cell clones. A pregnancy recognition signal, interferon tau (IFNτ), is a potential marker to qualify trophectoderm cells in ungulates. Aggregation of clone embryos is one of the methods to improve cloning efficiency in mouse (Boiani et al. 2003 EMBO J. 22, 5304–5312). Here, we evaluated the outcome of aggregating bovine clones using the bioactivity of IFNτ produced by clone blastocyst outgrowths. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39°C in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂. Aggregation of embryos was performed at the 8–16-cell stage by removing the zonae pellucidae with 0.5% pronase and placing 2 zona-free embryos into a microwell on the bottom of a culture dish. Embryos were cultured until Days 7–8. For outgrowth culture, blastocysts at Days 7–8 were individually placed on 4-well or 24-well plates containing mitotically inactivated STO feeder layers, and cultured at 37°C under 5% CO₂ in air in 1 mL of DMEM with 10% FBS in each well. The medium from each well (1 mL) was collected for analysis and replaced at Day 7, 14, and then every 3 days until 50 days. IFNτ antiviral assay was performed as described previously (Talbot et al. 2000 Biol. Reprod. 62, 235–247). IFNτ activity of outgrowths derived from clone aggregates was compared with those from single clones and IVF embryos. The number of IFNτ-positive outgrowths from single and aggregate IVF embryos decreased gradually until Day 32 and Day 35, respectively. In contrast, positive outgrowths from both single and aggregate clone embryos decreased dramatically before Day 26. While all of the positive IVF outgrowths at Day 35 were still positive at Day 50, the number of positive clone outgrowths decreased after Day 35. At Day 50, 66.7% (4 of 6), 42.9% (3 of 7), 11.8% (2 of 17), and 0% (0 of 18) of outgrowths were positive for IFNτ activity in IVF singles, IVF aggregates, clone singles, and clone aggregates, respectively, consistent with the typical loss of somatic cell clone fetuses during the first trimester and development rates to term. We conclude that IFNτ bioactivity adequately reflected the in vivo development of clones vs. IVF embryos, but that clone–clone aggregation had no quantitative benefit on extended in vitro culture.