Selected Amino Acids Promote Mouse Pre-implantation Embryo Development in a Growth Factor-Like Manner

Abstract

Groups of amino acids, and some selected amino acids, added to media used for culture of pre-implantation embryos have previously been shown to improve development in various ways including survival to the blastocyst stage, increased blastocyst cell number and improved hatching. In this study, we cultured 1-cell mouse embryos for 5 days to the hatching blastocyst stage in isosmotic medium (270 mOsm/kg) at high density (10 embryos/10 µL), where autocrine/paracrine support of development occurs, and low density (1 embryo/100 µL), where autocrine/paracrine support is minimized and development is compromised. When 400 µM L-Pro or 1 mM L-Gln was added to embryos at low density, the percentage of embryos reaching the blastocyst stage and the percentage hatching increased compared to low-density culture without these amino acids, and were now similar to those for embryos cultured at high density without amino acids. When L-Pro or L-Gln was added to embryos at high density, the percentage of embryos reaching the blastocyst stage didn't change but hatching improved. Neither embryo culture density nor the presence of these amino acids had any effect on blastocyst cell number. D-Pro and the osmolytes Gly and Betaine did not improve embryo development in low-or high-density culture indicating the mechanism was stereospecific and not osmotic, respectively. L-Pro-and L-Glnmediated improvement in development is observed from the 5-cell stage and persists to the blastocyst stage. Molar excess of Gly, Betaine or L-Leu over L-Pro eliminated improvement in development and hatching consistent with them acting as competitive inhibitors of transporter-mediated uptake across the plasma membrane. The L-Pro effect is dependent on mTORC1 signaling (rapamycin sensitive) while that for L-Gln is not. The addition of L-Pro leads to significant nuclear translocation of p-Akt S473 at the 2-and 4-cell stages and of p-ERK1/2 T202/Y204 nuclear translocation at the 2-, 4-, and 8-cell stages. L-Pro improvement in embryo development involves mechanisms analogous to those seen with Pro-mediated differentiation of mouse ES cells, which is also stereoselective, dependent on transporter uptake, and activates Akt, ERK, and mTORC1 signaling pathways.

Different response of embryos originating from control and obese mice to insulin <i>in vitro</i>

“…In the derived blastocysts, the cell proliferation, differentiation and death rates were determined using morphological fluorescence staining. To evaluate the possible mechanisms of different responses to insulin, mRNA expression of insulin receptor, insulin-like growth factor I receptor, and selected genes representing two main branches of insulin receptor signaling (phosphatidylinositol 3-kinases [PI3Ks] and mitogen-activated protein kinases [MAPKs] [ 21 , 22 , 23 ]) were analyzed in the blastocysts recovered from control and obese dams using real-time RT-PCR.…”

Non-Invasive Human Embryo Metabolic Assessment as a Developmental Criterion

“…The significantly high release of Ala and Gln is consistent with Houghton et al [ 5 ], and inconsistent with Sturmey et al, who declared that mouse embryos at blastocyst stages consumed Gln at a significantly higher rate than the early cleavage stages [ 8 ]. L-Gln is considered to be an essential component of culture media during embryo development over the 2- to 8-cell stages in a number of species [ 24 , 27 ]. It can be metabolized to other AAs (i.e., Glu, Ala, and Asp) [ 28 ] and ATP through the TCA cycle, synthesizes de-novo purine and pyrimidine, protects the embryo against oxidative stress, and acts as an organic osmolyte and a putative regulator in Glc metabolism [ 8 ].…”

“…Gly, a critical AA for the preimplantation development of both early and late-stage embryos [ 29 ] was also released to the media. Gly is abundant in oviduct fluid [ 29 ], and has been reported to be an essential precursor of polypeptide and nucleic acid synthesis, one of the key organic osmolytes and heavy metal chelators, which undergoes less apoptosis in Gly-cultured embryos [ 8 , 27 , 30 , 31 ]. Tracking the fate of orally administered 15 N-Gly showed that the Gly nitrogen group is mainly transferred to Ser, urea, Gln/Glu, Ala, and other AAs (Leu, Ile, Val, Orn, Pro, and Met) [ 32 ].…”

The Multifaceted Roles of Proline in Cell Behavior

“…Supplemental L-Pro (50-250 µM) improves proliferation of ESCs (Figure 8; Washington et al, 2010;Casalino et al, 2011), development of pre-implantation embryos (Morris et al, 2020) and fetus survival (Liu et al, 2019). L-Pro is internalized into stem cell cytoplasm through the SLC38A2 (SNAT2) transporter (Tan et al, 2011), and halofuginone (prolyl-tRNA synthetase inhibitor) fully counteracts L-Pro induction of cell proliferation (D'Aniello et al, 2015).…”