Reproduction Abstracts

The composition of the external environment can have a significant impact on intracellular signaling pathways during early embryonic development. Components of the culture media, especially amino acids, are known to improve embryo development. We have shown that L-proline and L-glutamine improve development to the blastocyst stage by acting in a growth factor-like way. The aim of this study is to investigate the effect of amino acids on signaling pathways in the embryo.

Zygotes were cultured in the presence or absence of either all 20 amino acids, or L-pro alone, for between 15 min and 48 h and collected for western blotting and immunofluorescence. Rapamycin (10 nM) was used to investigate the role of the mTOR pathway in the developmental effects of amino acids.

Culture of embryos in the presence of rapamycin prevented the L-pro-induced improvement in development, suggesting mTORC1 activity is up-regulated by L-pro. However, western blotting did not show any change in phosphorylation of 4E-BP1 or rpS6, which are downstream of mTORC1, following treatment with L-pro for 48 h. Similarly, L-pro had no effect on activity of ERK1/2 or AKT pathways. However, the presence of all 20 amino acids for 6 h increased the phosphorylation of AKT and ERK1/2 in a cell cycle dependent way. These results indicate that L-pro-mediated developmental improvement in cultured embryos occurs via signaling pathways, including activity that may be cell cycle dependent and independent.