112 Birc6/Apollon INVOLVED IN BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT

Abstract

Baculoviral inhibitor of apoptosis repeat-containing (Birc)6 gene is known by different names as Birc6, Apollon, Bruce, or KIAA1289. The bovine Birc6 contains the ubiquitin conjugating enzyme. A deletion of the C-terminal half and the ubiquitin conjugating enzyme domain of the mouse apollon is known to activate apoptosis and resulted in embryonic lethality. Moreover, its overexpression in drug-resistance cancer cells suggested its antiapoptotic role. However, its involvement in bovine preimplantation embryo development is not yet known. Therefore, this experiment was conducted to investigate the role of Birc6 in bovine preimplantitive embryos by targeted suppression of the Birc6 mRNA using RNA interference (RNAi). For this, gene-specific long double-stranded RNA (dsRNAi) and short hairpin RNA (shRNAi) targeting Birc6 were in vitro synthesized. In vitro-fertilized zygotes were injected with dsRNAi (n = 347) and shRNAi (n = 374), and uninjected zygotes (n = 366) were used as a control. Sham injection was not used as an additional control in this experiment, because water-injected and uninjected control embryos (Nganvongpanit et al. 2006 Mol. Reprod. Dev. 53, 153–163) have shown similar embryonic development. Following microinjection, the proportion of uncleaved zygotes, 2-, 4- and 8-cell embryos, and the Birc6 mRNA knockdown were determined at 96 h post-insemination (hpi). The effect of Birc6 knockdown on genes involved in apoptotic pathway was evaluated by determining the pro-apoptotic Bax mRNA. The specificity of RNAi was investigated by quantifying glyceraldehyde 3-phosphate dehydrogenase in each treatment group. SPSS version 12.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The least significant difference t-test pairwise comparison was done between the treatment groups to test varation in uncleaved zygotes, 2-, 4-, and 8-cell embryos. The results showed that the proportion of 8-cell embryos (mean ± SEM) was significantly (P < 0.05) higher in control (49.1 ± 3.3) compared to dsRNAi (34 ± 4.4) and shRNAi (33.5 ± 5.2) groups, but the proportion of the 2-cell embryos was significantly higher in dsRNAi (15.1 ± 2.3) and shRNAi (14.9 ± 3.5) compared to control (8.9 ± 1.7), indicating developmental arrest of 2-cell embryos in RNAi groups before reaching the 8-cell stage. Furthermore, the proportion of uncleaved embryos tended to be higher in dsRNAi (34.6 ± 3.2) and shRNAi (35.2 ± 4.4) compared to control (28.2 ± 3.02). The data on the level of suppression on these embryos showed that the Birc6 mRNA in 2-cell embryos was reduced by 85% in dsRNAi and by 50% in shRNAi treatment groups. In 8-cell embryos, 40 and 50% knockdown was achieved in shRNAi and dsRNAi groups, respectively, compared to the control. Similarly, in 8-cell embryos, there was a 23 and 70% increase in mRNA level of Bax in dsRNAi and shRNAi groups, respectively, compared to control, indicating the incidence of apoptosis in the RNAi groups. In conclusion, significantly lower proportions of 8-cell embryos and higher proportion of 2-cell and uncleaved embryos in Birc6 RNAi treatment groups compared to the control groups may reveal the involvement of Birc6 in bovine preimplantation embryo development.