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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.)

Embryonic genome activation (EGA) is the first major step towards successful initiation of preimplantation development, which culminates in the formation of implantation-competent embryos. EGA occurs at species-specific embryonic cell stages. In the present work, EGA was identified for buffalo embryos by studying the development rate of embryos in normal as well as imposed transcription block conditions, analyzing bromo-uridine triphosphate (BrUTP) incorporation rates as evidence of de novo transcription initiation, and studying the expression status of eukaryotic translation initiation factor 1A (eIF1A), U2 auxiliary splicing factor (U2AF), and polyadenylate polymerase (PAP) genes at different embryonic cell stages. Under normal, in vitro fertilization and culture conditions, about 26% and 17% of oocytes could reach morula and blastocyst stages, respectively, but no embryos could progress beyond 8-cell stages in presence of α-amanitin. Culturing embryos in the presence of BrUTP revealed a marked increase in its incorporation between 4- and 8-cell stages. All genes studied displayed an abrupt increase in expression between 4- and 8-cell stages; PAP expression was upregulated earlier from 2- to 4-cell stages. About 65% of PAP transcripts from the 4-cell stage and more than 70% of eIF1A, U2AF, and PAP transcripts at 8-cell stage embryos were found to be synthesized de novo. Together, these data suggest that a minor EGA in buffalo embryos happens from 2- to 4-cell stages, while the major EGA takes place from 4- to 8-cell stage transition., (Copyright © 2012 Wiley Periodicals, Inc.)

HOX proteins are transcription factors that play a major role in patterning the body axis of vertebrates from the gastrulation stage. While nothing has been reported so far about their roles at earlier stages, there is evidence that some HOX genes are expressed before gastrulation. The objective of this work was to study the pattern of expression of several HOX genes during oocyte maturation and early embryonic development up to the blastocyst stage. Using nested PCR, HOXD1, HOXA3, HOXD4, HOXB7, HOXB9, and HOXC9 transcripts were detected in bovine oocytes and early embryos at various frequencies depending on the stage of development. Quantitative PCR was performed on bovine oocytes and early embryos: relative expression of HOXD1, HOXA3, and HOXC9 decreased sharply after the 5-8 cell stage. HOXB9 relative expression increased between the oocyte and the morula stage. All transcripts seemed to be of maternal origin before the maternal to embryonic transition, as demonstrated by blocking transcription with α-amanitin. Reverse transcription was performed with either hexamers or oligo-dT, allowing for the determination that HOXC9 transcripts were slightly deadenylated during oocyte maturation; HOXD1, HOXA3, and HOXB9 transcripts were not, indicating that they could be translated. Hoxd1, Hoxa3, Hoxb9, and Hoxc9 expression was also detected in mouse oocytes and early embryos. A similar pattern of expression was found in the two species. In conclusion, mammalian HOX genes might be implicated in the control of oocyte maturation, the maternal-to-embryonic transition or the first steps of embryo differentiation., (Copyright © 2011 Wiley-Liss, 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.

We have investigated the effect of alpha-amanitin treatment of mouse blastocysts on their further development. Twenty-four-hour treatments with alpha-amanitin at 1 microg/ml do not interfere with embryo survival or gross morphological development over the next few days although the rate of synthesis of polyA-containing RNA is dramatically reduced during exposure to the antimetabolite. Effects upon rates of protein synthesis are not observed initially, but incorporation of (35S)methionine into acid-insoluble material is ultimately inhibited by approximately 50% in all the treated samples. Developmental markers do not all respond in the same way to the alpha-amanitin treatments used. Whereas levels of beta-glucuronidase and plasminogen activator are virtually unaffected by the antimetabolite, alpha-amanitin treatment interferes with delta5,3beta-hydroxysteroid dehydrogenase activity in trophoblast cells and formation of a bilayered inner cell mass. The effects upon the latter markers differ quantitatively with the various exposure periods to alpha-amanitin. On the basis of these observations, we propose that mRNAs for at least some developmental markers in mouse blastocysts are synthesized long before they are translated.