Abstract
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.
Highlights
The presence of individual and groups of amino acids in embryo culture medium impacts preimplantation embryo development [1,2,3,4,5,6,7,8]
The percentage of embryos that had compacted at 72 h and developed to the blastocyst stage at 120 h following in vitro fertilisation (IVF) in Pro increased compared to oocytes fertilised in the absence of AA
PROT and PAT1/2 Are Expressed in Oocytes We investigated the expression of three Pro transporters with similar amino acid uptake profiles to what we observed in the oocyte
Summary
The presence of individual and groups of amino acids in embryo culture medium impacts preimplantation embryo development [1,2,3,4,5,6,7,8]. The addition of specific amino acids or groups of amino acids to the IVF medium can improve various aspects of subsequent development, including the percentage of embryos that reach the blastocyst stage and hatch [9,10], indicating that amino acids can enter the oocyte in sufficient quantities during the period of fertilisation to exert effects on later embryo development. When zygotes are cultured at low density (1 embryo/100 μL, to eliminate the action of embryo-derived growth factors), the addition of Pro increases both the number of embryos reaching the blastocyst stage and their hatching by stimulating development within the late two-cell to eight-cell stages [7]. The addition of Pro promotes differentiation and neural lineage commitment in embryonic stem cell (ESC) models of post-implantation development [11,12]
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