Involvement of SMAD Signaling in Bovine Early Embryonic Development.

Abstract

We have previously demonstrated a positive functional role for maternal (oocyte-derived) follistatin in control of time to first cleavage of bovine embryos, subsequent development to the blastocyst stage and in blastocyst cell allocation to the trophectoderm lineage. Follistatin binds to and inhibits activity of members of the transforming growth factor superfamily, whose signals are mediated intracellularly primarily through activation of receptor associated SMADs (rSMADs) including SMAD 2/3 (activin, TGF-beta) and SMAD 1/5/8 (BMPs). Thus, we hypothesized that follistatin's positive actions on early embryogenesis may be mediated via inhibition of rSMAD signaling. To address this hypothesis we determined the temporal expression and effects on bovine early embryogenesis of siRNA mediated knockdown of SMAD 4, a common component of above signaling pathways which complexes with respective rSMADs. Temporal changes in SMAD 4 mRNA during early embryogenesis were determined by real time PCR analysis of embryos collected at pronuclear, 2-, 4-, 8- and 16-cell and morula and blastocyst stages. SMAD 4 mRNA abundance was highest in 2-cell embryos and decreased dramatically to very low levels between the 8- to 16-cell stages, suggesting that SMAD 4 transcripts in early embryos are oocyte-derived. The maternal origin of SMAD 4 transcripts was confirmed by real time PCR analysis of embryos cultured to the 8-cell stage in the presence of the transcription inhibitor alpha-amanitin. Furthermore, SMAD 4 mRNA was higher in alpha-amanitin treated embryos, suggesting potential transcriptional dependence of observed decline in maternal SMAD 4 mRNA. To determine the functional role of SMAD 4 in bovine early embryogenesis, multiple siRNA species targeting bovine SMAD 4 were synthesized and evaluated for efficacy in reducing SMAD 4 mRNA levels (in 4-cell embryos) following microinjection into presumptive zygotes. Two of the validated SMAD 4 siRNA species with > 90 % efficiency in targeting SMAD 4 mRNA were combined and the efficacy of this siRNA cocktail (utilized in all subsequent studies) in reducing SMAD 4 mRNA and protein in early embryos verified. Presumptive zygotes were then injected with SMAD 4 siRNA, a negative control siRNA or served as uninjected controls. Effects of SMAD 4 knockdown on proportion of embryos cleaving early (within 30 h post insemination; hpi), total cleavage rates (determined 48 hpi), proportion of embryos developing to 8-16 cell (72 hpi) and blastocyst stages (d 7) and on blastocyst CDX2 mRNA abundance (index of trophectoderm cell allocation) were determined. SMAD 4 knockdown significantly reduced the proportion of embryos that cleaved early and embryonic development to 8- to 16-cell and blastocyst stages relative to uninjected and negative control siRNA injected embryos. Such results support a functional role for SMAD signaling in control of time to first cleavage and subsequent development to the blastocyst stage. However, effects of SMAD 4 knockdown on abundance of CDX2 mRNA in resulting blastocysts were not observed. Collectively, results support a requirement for the common SMAD (SMAD 4) of maternal origin in promoting bovine early embryogenesis, but do not suggest that previously observed stimulatory effects of follistatin on embryonic development are likely mediated via blocking stimulation of classical SMAD 4 dependent rSMAD signaling via endogenous TGF-beta superfamily members. (poster)