63 INTERFERON-TAU EXPRESSION FROM PRIMARY TROPHECTODERM OUTGROWTHS OF BOVINE BLASTOCYSTS: COMPARISON BETWEEN IVP, NT, AND PARTHENOGENIC EMBRYOS

Abstract

Interferon-tau (IFN-tau) is expressed soon after bovine blastocyst formation and might be useful as a marker of appropriate biological function in embryos produced by nuclear transfer. To assess this possibility we have compared IFN-tau levels in the conditioned medium of primary trophectoderm cultures derived from IVP, nuclear transfer (NT), or parthenogenic bovine embryos. Embryos were produced from in vitro-matured cumulus-oocyte complexes processed from local slaughterhouse ovaries or obtained from Bomed, Inc. (Madison, WI, USA). In vitro fertilization, NT, and parthenogensis were as previously described (Talbot et al., 2000 Tissue and Cell, 32, 9–27) except that embryo culture was in G1/G2 medium in 5% oxygen (Lane et al., 2003 Theriogenology, 60, 407–419). Each 8–11-day embryo was cultured individually in a 4-well plate well (Nunc) with STO feeder cells using DMEM medium containing 10% fetal bovine serum as previously described (Talbot et al., 2000 Biol. Reprod. 62, 235–247). Any contaminating epiblast or endoderm was physically dissected and discarded so as to produce pure trophectoderm outgrowths. The success/failure ratio for colony formation was similar for IVP and NT embryos (IVP = 155/29; NT = 104/25), but was significantly different (P < 0.05) for parthenogenic embryos (54/43). Trophectoderm colonies reached diameters of 1 to 1.5 cm in 3–4 wk, and, at this time, 72-h-conditioned cell culture medium was harvested, frozen, and measured for IFN-tau anti-viral activity as previously described (Talbot et al., 2000 Biol. Reprod. 62, 235–247). From 313 observations, IFN-tau production was analyzed as a two-factor mixed linear model. Differences in IFN-tau production by type of embryo were statistically significant (F = 42.61; P < 0.0001; df = 2). Mean comparisons were done with Sidak adjusted P-values so that the experiment-wise error was 0.05. IFN-tau production means for IVP-, NT-, and parthenogenic-derived trophectoderm were 4311 IU mL⁻¹ (n = 155), 626 IU mL⁻¹ (n = 104), and 1595 IU mL⁻¹ (n = 54), respectively. The results show that mean IFN-tau production from trophectoderm cultures derived from NT embryos is significantly reduced in comparison to IVP- and parthenogenote-derived cultures. Parthenogenote-derived cultures also produced significantly less IFN-tau than IVP embryos on average. IFN-tau production from trophectoderm outgrowths may be a useful measure of NT reprogramming success.