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mTORC1/2 signaling is downregulated by amino acid-free culture of mouse preimplantation embryos and is only partially restored by amino acid readdition.

American journal of physiology. Cell physiology (2020-10-15)
Radu C Zamfirescu, Margot L Day, Michael B Morris
ABSTRACT

Development of the mammalian preimplantation embryo is influenced by autocrine/paracrine factors and the availability of nutrients. Deficiencies of these during in vitro culture reduce the success of assisted reproductive technologies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway integrates external and internal signals, including those by amino acids (AAs), to promote normal preimplantation development. For this reason, AAs are often included in embryo culture media. In this study, we examined how withdrawal and addition of AAs to culture media modulate mTORC1 pathway activity compared with its activity in mouse embryos developed in vivo. Phosphorylation of signaling components downstream of mTORC1, namely, p70 ribosomal protein S6 kinase (p70S6K), ribosomal protein S6, and 4E binding protein 1 (4E-BP1), and that of protein kinase B (Akt), which lies upstream of mTORC1, changed significantly across stages of embryos developed in vivo. For freshly isolated blastocysts placed in vitro, the absence of AAs in the culture medium, even for a few hours, decreased mTORC1 signaling, which could only be partially restored by their addition. Long-term culture of early embryos to blastocysts in the absence of AAs decreased mTORC1 signaling to a greater extent and again this could only be partially restored by their inclusion. This failure to fully restore is probably due to decreased phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC2 signaling in culture, as indicated by decreased P-AktS473. mTORC2 lies upstream of mTORC1 and is insensitive to AAs, and its reduced activity probably results from loss of maternal/autocrine factors. These data highlight reduced mTORC1/2 signaling activity correlating with compromised development in vitro and show that the addition of AAs can only partially offset these effects.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
L-Glutamine, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
Sigma-Aldrich
Mineral oil, light oil, BioReagent, suitable for mouse embryo cell culture
Sigma-Aldrich
Poly(vinyl alcohol), 87-90% hydrolyzed, average mol wt 30,000-70,000
Sigma-Aldrich
L-Proline, from non-animal source, meets EP, USP testing specifications, suitable for cell culture
Sigma-Aldrich
Hyaluronidase from sheep testes, Type II, lyophilized powder, ≥300 units/mg solid