In Vitro Fertilisation of Mouse Oocytes in L-Proline and L-Pipecolic Acid Improves Subsequent Development

Exposure of oocytes to specific amino acids during in vitro fertilisation (IVF) improves preimplantation embryo development. Embryos fertilised in medium with proline and its homologue pipecolic acid showed increased blastocyst formation and inner cell mass cell numbers compared to embryos fertilised in medium containing no amino acids, betaine, glycine, or histidine. The beneficial effect of proline was prevented by the addition of excess betaine, glycine, and histidine, indicating competitive inhibition of transport-mediated uptake. Expression of transporters of proline in oocytes was investigated by measuring the rate of uptake of radiolabelled proline in the presence of unlabelled amino acids. Three transporters were identified, one that was sodium-dependent, PROT (SLC6A7), and two others that were sodium-independent, PAT1 (SLC36A1) and PAT2 (SLC36A2). Immunofluorescent staining showed localisation of PROT in intracellular vesicles and limited expression in the plasma membrane, while PAT1 and PAT2 were both expressed in the plasma membrane. Proline and pipecolic acid reduced mitochondrial activity and reactive oxygen species in oocytes, and this may be responsible for their beneficial effect. Overall, our results indicate the importance of inclusion of specific amino acids in IVF medium and that consideration should be given to whether the addition of multiple amino acids prevents the action of beneficial amino acids.