Effect of pre-implantation maternal low protein diet on embryos and embryoid bodies
mesheuropmc: embryonic structures
Previous studies have shown that poor maternal nutrition during pregnancy may induce metabolic syndrome in adults. In this study, we explored the mechanisms of how maternal low protein diet during the first 3.5 days of pregnancy, programs embryo development and embryonic stem cell function. Our results showed that mouse maternal low protein diet (9% casein; Emb-LPD) enhanced both Clathrin dependent and independent endocytosis in both in vivo blastocysts and in vitro differentiated embryoid bodies (EB day 5) compared with maternal normal protein diet (18% casein; NPD) controls. This increase in endocytosis was accompanied by an increase in lysosome volume per cell. This was done by confocal microscopy and 3D image analysis. To determine whether this effect on the lysosome system was due to autophagy or simply due to increased endocytosis, we studied the expression of LC3 protein, Clathrin, Megalin (also named as low density lipoprotein receptor 2 (LRP2)) and Cubilin. Immunostaining and Western blot analysis revealed Megalin and Cubilin were significantly up-regulated in Emb-LPD embryos and EBs, whilst Clathrin protein level was marginally increased and LC3 protein unaltered. This enhanced nutrient uptake ability was maintained even after cells or embryos were re-introduced into a normal environment in vitro. Thus, stimulated nutrient uptake in day 5.5 EB showed compensatory growth, known to associate with long-term disease symptoms. To understand the mechanisms involved, we investigated elements of the mTOR pathway. In vitro culture of early embryos in the presence of reduced levels of the three branched-chain amino acids (Lecine, Valine and Isoleucine) as occurring in Emb-LPD uterine fluid resulted in stimulated endocytosis of Trophectoderm (TE). In addition, we found although mTORC1 was partially suppressed, mTORC2 downstream RhoA-Actin interaction was stimulated in blastocysts by observing more actin and RhoA protein in Emb-LPD blastocysts as well as that inhibiting RhoA function abolished the enhanced endocytosis by Emb-LPD. We also investigated epigenetic changes induced by Histone deacetylase 3 (Hdac3) in terms of regulation of genes involved in Extraembronic Endoderm (XEN) differentiation and cardiomyocyte differentiation. We found that Emb-LPD EBs expressed reduced Gata6 and exhibited increased histone deacetylation at promoter of Gata6, together with increased Hdac3 expression. Our results reveal for the first time at the cellular level how early embryos respond to poor nutrition environment and reprogram to protect fetal growth. This further helps us to understand the mechanism of how adult metabolic syndrome can be originated from environment which early embryos were exposed to.
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