167 EXPRESSION OF INTERFERON τ IN BOVINE EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION

Abstract

Parthenogenetic activation (PA) is a useful technique for reproductive technologies such as somatic cell nuclear transfer. Furthermore, there is a possibility of embryonic stem cell establishment deriving from PA embryos (Cibelli et al. 2002 Science 295, 819). Understanding of the ability of development and differentiation in PA embryos is important for the application. This study was designed to assess the gene expression of octamer-binding transcription factor (OCT-4) and interferon τ (IFNτ) in bovine PA embryos at the blastocyst (BC) and elongated (EL) stages, and the protein secretion of IFNτ at the EL stage. PA embryos were produced from oocytes matured in vitro (24 h), activated with Ca-ionophore (5 min) and electric pulse, and then treated with cytochalasin B and cycloheximide (5 h). In vivo-produced (Vivo) embryos were obtained non-surgically at Day 8 (Day 0 = estrus) from superovulated donor cows. PA or Vivo embryos were transferred to recipient cows (PA: 10 embryos/cow; Vivo: 1 embryo/cow) at Day 8, and then recovered non-surgically with uterine flushings at Day 16. Total RNA in single BC and EL embryos were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. IFNτ secretion of uterine flushings was estimated by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). The cleavage and blastocyst developmental rates of PA oocytes were 65.8 and 29.7%, respectively. Most embryos had positive signals of OCT-4 and IFNτ regardless of the origin and stage of embryos. The relative abundance (mean ± SEM) of OCT-4 expression in PA and Vivo embryos dropped to its lowest level (1.78 ± 0.32 and 1.14 ± 0.45, respectively) at the EL stage, and it was significantly lower than that at the BC stage (1018.87 ± 148.69 and 696.29 ± 151.80, respectively; P < 0.05). The transcript level of OCT-4 was not significantly different between PA and Vivo embryos at both stages. Although the transcript level of IFNτ in PA and Vivo embryos increased significantly at the EL stage (0.36 ± 0.06 and 7.68 ± 2.01, respectively) from the BC stage (0.03 ± 0.01 and 0.01 ± 0.004, respectively; P < 0.05), that in PA embryos was significantly lower than that in Vivo embryos at the EL stage (P < 0.01). The total amount (mean ± SEM) of IFNτ in uterine flushings from cows with transferred PA embryos was 3.38 ± 0.35 µg (the number of embryos in each uterine flushing was unknown), and it was low compared with that from cows with Vivo embryos (13.40 ± 3.03 µg). Our results indicate that bovine PA embryos have the ability to secrete IFNτ in the uteri of recipient cows at the EL stage, and there is a similar expression pattern of OCT-4 for Vivo embryos.