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Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent., (© 2014 by the Society for the Study of Reproduction, Inc.)

The early bovine preimplantation embryo is resistant to proapoptotic signals until around the 8- to 16-cell stage. We hypothesized that 2-cell embryos have higher amounts of antiapoptotic proteins and lower amounts of proapoptotic proteins when compared to embryos ≥16 cells. Steady-state concentrations of mRNA for the antiapoptotic genes BCL2 and HSPA1A were higher for MII oocytes, 2-cell embryos, and 2-cell embryos treated with alpha-amanitin as compared to ≥16-cell embryos. Steady-state concentrations of mRNA for the proapoptotic gene BAD increased in embryos ≥16 cells. There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1, DFFA, or BAX. Using immunohistochemistry, it was found that BCL2 was present in greater relative concentrations for 2-cell embryos than for embryos ≥16 cells. These results were confirmed by Western blotting. Relative amounts of immunoreactive BAX detected by immunofluorescence were lower for 2-cell embryos than for embryos ≥16 cells. Using Western blotting, a high molecular weight (46 kDa) form of BAX was highest in ≥16-cell embryos, intermediate in 2-cell embryos, and lowest in MII oocytes. There were no effects of stage of development on relative amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence. Treatment of embryos with alpha-amanitin from Day 0 to Day 5 or Day 4 to Day 5 after insemination reduced activation of group II caspases and terminal deoxynucleotidyl transferase dUTP nick end labeling after treatment with the proapoptotic signal C(2) ceramide at Day 5 after fertilization. Thus, transcription of BAX or other proteins is required for acquisition of the capacity for apoptosis. Results support the idea that changes in amounts of BCL2 family members are important for the inhibition of apoptosis in the 2-cell embryo and in the establishment of the capacity for apoptosis later in development.