In vivo knockdown of FGFR2 and MET mRNAs in trophectoderm of ovine conceptuses retards their development via abrogation of MAPK and MTOR pathways

Abstract Text:

The paradigm of down-regulation of progesterone receptor in uterine epithelia prior to implantation is common to sheep, cow, pigs, rhesus monkey, women and mice. Therefore, progestamedins, which derive from PGR positive uterine stromal cells and include hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7) and FGF10, regulate uterine luminal (LE), superficial glandular (sGE) and glandular (GE) epithelia during the estrous cycle/early pregnancy. Previous studies with sheep demonstrated the existence of receptors for HGF (HGFR, encoded by MET), as well as FGF7 and FGF10 (FGFR2) in conceptus trophectoderm (Tr). However, the biological roles of progestamedins in conceptus development are unknown. In this study, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown of translation of FGFR2 and MET mRNAs in ovine conceptus Tr. Normality of data and homogeneity of variance were tested using the Shapiro-Wilk test and Brown-Forsythe test in SAS, respectively. Data were analyzed by least-squares one-way analysis of variance (ANOVA) and post hoc analysis (the Fisher least significant difference) with each ewe/conceptus as an experimental unit. P<0.05 was considered significant. Translational knockdown of MET mRNA severely retarded conceptus development whereas translational knockdown of FGFR2 mRNA resulted in small, thin and less elongated conceptus as compared with MAO control conceptuses. Both MAO-MET and MAO-FGFR2 conceptuses were functionally abnormal based on lower (P<0.05) production of interferon tau, the pregnancy recognition signal in sheep. Quantitative immunofluorescence (IF) analysis demonstrated that MAOs were evenly delivered (P>0.05) into Tr, but not uterine LE, sGE and GE, and that the abundance of both MET and FGFR2 proteins in Tr was decreased (P<0.01) by 83.3% and 93.3%, respectively. Western blot analysis using ovine Tr cells treated with respective MAOs also validated knockdown efficiencies of both MAO-MET and MAO-FGFR2. Further quantitative IF revealed that as compared with MAO control, active caspase-3 in Tr of MAO-MET and MAO-FGFR2 conceptuses was increased (P<0.01) by 4.5- and 6.5-fold, respectively. Moreover, phosphorylation of P38 was decreased (P<0.01) by 68.7% and 84.4% in MAO-MET and MAO-FGFR2, respectively, as compared to MAO control; whereas phosphorylation of MTOR was decreased (P<0.01) by 75.0% and 83.3% in MAO-MET and MAO-FGFR2 conceptuses, respectively. Interestingly, phosphorylation of TSC2 was decreased (P<0.01) by 79.2% in Tr of MAO-MET compared with MAO control, but was no different (P>0.05) between MAO-FGFR2 and MAO control conceptuses.  Collectively, these results demonstrate critical roles for progestamedins (i.e., HGF, FGF7 and FGF10) in ovine conceptuses that are mediated via their receptors (MET and FGFR2) and activation of MAPK and MTOR pathways, and that translational knockdown of MET and FGFR2mRNAs increased apoptosis and retarded conceptus development during early pregnancy. 

Keywords: Progestamedin, Trophectoderm, MTOR, MAPK, Sheep