The online version of this article (https://doi.org/10.1186/s12958-017-0318-6) contains supplementary material, which is available to authorized users.
TGF-β signaling pathways regulate several crucial processes in female reproduction. AKT is a non-SMAD signaling pathway regulated by TGF-β ligands essential for oocyte maturation and early embryonic development in the mouse, but its regulatory role in bovine early embryonic development is not well established. Previously, we demonstrated a stimulatory role for follistatin (a binding protein for specific members of TGF-β superfamily) in early bovine embryonic development. The objectives of the present studies were to determine the functional role of AKT signaling in bovine early embryonic development and embryotrophic actions of follistatin.
We used AKT inhibitors III and IV as pharmacological inhibitors of AKT signaling pathway during the first 72 h of in vitro embryo culture. Effects of AKT inhibition on early embryonic development and AKT phosphorylation were investigated in the presence or absence of exogenous follistatin.
Pharmacological inhibition of AKT signaling resulted in a significant reduction in early embryo cleavage, and development to the 8- to 16-cell and blastocyst stages (d7). Treatment with exogenous follistatin increased AKT phosphorylation and rescued the inhibitory effect of AKT inhibitors III and IV on AKT phosphorylation and early embryonic development.
Collectively, results suggest a potential requirement of AKT for bovine early embryonic development, and suggest a potential role for follistatin in regulation of AKT signaling in early bovine embryos.
Additional file 1: Figure S1. Effect of AKT inhibitors III and IV treatments on AKT-Ser473 phosphorylation levels in early bovine embryos. IVF embryos were cultured in presence of 0, 25, 50 or 75 μM AKT inhibitor III or 0, 1.5, 2.5 or 3.5 μM AKT inhibitor IV for 10 h, then subjected to Western blot for pAKT-Ser473, tAKT and actin analysis (n = 3 replicates/antibody, n = 20 embryos/treatment). Data were normalized relative to abundance of actin and phosphorylation levels (a, b) were expressed as pAKT/tAKT. Representative Western blot images are shown. Data are expressed as mean ± SEM. Values with different superscripts among treatments indicate significant differences (P < 0.05). (TIFF 650 kb)12958_2017_318_MOESM1_ESM.tif
Additional file 2: Figure S2. Effect of follistatin treatment on AKT-Ser473 phosphorylation in early bovine embryos. Presumptive zygotes were cultured with 0 or 10 ng/ml recombinant human follistatin in the presence or absence of AKT inhibitor III (75 μM) for 10 h (n = 5 replicates, n = 20 zygotes/group) (a), or Presumptive zygotes were cultured in the presence or absence of 10 ng/ml follistatin for 24 h (n = 5 replicates, n = 20 zygotes/group) (b). Samples were subjected to Western blot analysis for pAKT-Ser473, tAKT and Actin. Expression levels were normalized to the abundance of an endogenous control actin. Phosphorylation level was expressed as pAKT/tAKT. Data are expressed as mean ± standard error. Values with different superscripts among treatments indicate significant differences (P < 0.05). Representative Western blots are shown. (TIFF 542 kb)12958_2017_318_MOESM2_ESM.tif
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- Functional role of AKT signaling in bovine early embryonic development: potential link to embryotrophic actions of follistatin
Sandeep K. Rajput
Joseph K. Folger
Jason G. Knott
Nabil A. Hemeida
Omaima M. Kandil
Refaat S. Ragab
George W. Smith
- BioMed Central
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