The inclusion of amino acids in pre-implantation embryo culture media is known to improve the rate of development and embryo viability. Our studies have shown that embryos cultured in L-proline from the zygote to the blastocyst stage develop better than embryos cultured in the absence of amino acids. This study aimed to identify the pre-implantation stage(s) at which L-proline transport is required for development to be improved and then to characterise the amino acid transporter(s) responsible for L-proline uptake into the embryo.
Mouse embryos were cultured in medium containing 400 mM L-proline and scored for development. Competitive substrates, immunostaining and embryos from SLC6A19 null mice were used to identify L-proline transporters in early embryos.
Embryos cultured with L-proline from the 2-cell stage developed better than embryos cultured with L-proline in earlier stages, suggesting that the later stages are crucial for L-prolines transport and subsequent effect. The presence of excess L-leucine or glycine in the medium prevented the improvement in development induced by L-proline, implicating the transporters SLC6A19 and SNAT2 since they transport L-proline, L-leucine and glycine. Immunostaining showed that SLC6A19 was present in the pre-implantation embryo and that it appeared to be expressed in the membrane from the 8-cell stage onwards. SLC6A19 null mice showed poor development to the BL stage, which was not improved by addition of L-proline to the medium. These results suggest that SLC6A19 may be responsible for L-proline uptake in the pre-implantation embryo and for the subsequent improvement in embryo development.
02 Sep 2014 - 04 Sep 2014