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The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo development, increasing the percentage that develop to the blastocyst stage and hatch. Staining of 2-cell and 4-cell embryos with tetramethylrhodamine methyl ester and 2′,7′-dichlorofluorescein diacetate showed that the culture of embryos in the presence of Pro, or either of these analogues, reduced mitochondrial activity and reactive oxygen species (ROS), respectively, indicating potential mechanisms by which embryo development is improved. Inhibition of the Pro metabolism enzyme, proline oxidase, by tetrahydro-2-furoic-acid prevented these reductions and concomitantly prevented the improved development. The ways in which Pro, PA and L4T reduce mitochondrial activity and ROS appear to differ, despite their structural similarity. Specifically, the results are consistent with Pro reducing ROS by reducing mitochondrial activity while PA and L4T may be acting as ROS scavengers. All three may work to reduce ROS by contributing to the GSH pool. Overall, our results indicate that reduction in mitochondrial activity and oxidative stress are potential mechanisms by which Pro and its analogues act to improve pre-implantation embryo development. [ABSTRACT FROM AUTHOR]

L-proline (Pro) has previously been shown to support normal development of mouse embryos. Recently we have shown that Pro improves subsequent embryo development when added to fertilisation medium during in vitro fertilisation of mouse oocytes. The mechanisms by which Pro improves embryo development are still being elucidated but likely involve signalling pathways that have been observed in Pro-mediated differentiation of mouse embryonic stem cells. In this study, we show that B0AT1, a neutral amino acid transporter that accepts Pro, is expressed in mouse preimplantation embryos, along with the accessory protein ACE2. B0AT1 knockout (Slc6a19−/−) mice have decreased fertility, in terms of litter size and preimplantation embryo development in vitro. In embryos from wild-type (WT) mice, excess unlabelled Pro inhibited radiolabelled Pro uptake in oocytes and 4–8-cell stage embryos. Radiolabelled Pro uptake was reduced in 4–8-cell stage embryos, but not in oocytes, from Slc6a19−/− mice compared to those from WT mice. Other B0AT1 substrates, such as alanine and leucine, reduced uptake of Pro in WT but not in B0AT1 knockout embryos. Addition of Pro to culture medium improved embryo development. In WT embryos, Pro increased development to the cavitation stage (on day 4); whereas in B0AT1 knockout embryos Pro improved development to the 5–8-cell (day 3) and blastocyst stages (day 6) but not at cavitation (day 4), suggesting B0AT1 is the main contributor to Pro uptake on day 4 of development. Our results highlight transporter redundancy in the preimplantation embryo. [ABSTRACT FROM AUTHOR]

The viability of embryos cultured in vitro is poor compared to those that develop in vivo. The lack of maternally derived growth factors in vitro may contribute to this problem. Insulin-like growth factor binding protein 3 (IGFBP3) is one such growth factor that has been identified in the maternal reproductive system. This study examined the role of autocrine and exogenous IGFBP3 in mouse preimplantation embryos. Embryos expressed IGFBP3 across all stages of preimplantation development, and addition of exogenous IGFBP3 to embryo culture media increased the rate of development to the 2-, 4-, 5-, and 8-cell stages. Addition of inhibitors of the IGF1 and EGF receptors prevented this IGFBP3-mediated improvement in developmental rate, but the effect was not cumulative, indicating that both receptors are transactivated downstream of IGFBP3 as part of the same signalling pathway. Acute exposure to IGFBP3 increased phosphorylation of Akt and rps6 in 4–8 cell embryos, suggesting activation of the PI3-kinase/Akt pathway downstream of the IGF1 and EGFR receptors to promote cell proliferation and survival. In conclusion, addition of IGFBP3 to embryo culture media increases early cleavage rates independent of IGF1 signalling and therefore, IGFBP3 addition to IVF culture media should be considered. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

L-proline (Pro) has previously been shown to support normal development of mouse embryos. Recently we have shown that Pro improves subsequent embryo development when added to fertilisation medium during in vitro fertilisation of mouse oocytes. The mechanisms by which Pro improves embryo development are still being elucidated but likely involve signalling pathways that have been observed in Pro-mediated differentiation of mouse embryonic stem cells. In this study, we show that B 0 AT1, a neutral amino acid transporter that accepts Pro, is expressed in mouse preimplantation embryos, along with the accessory protein ACE2. B 0 AT1 knockout ( Slc6a19 -/- ) mice have decreased fertility, in terms of litter size and preimplantation embryo development in vitro. In embryos from wild-type (WT) mice, excess unlabelled Pro inhibited radiolabelled Pro uptake in oocytes and 4-8-cell stage embryos. Radiolabelled Pro uptake was reduced in 4-8-cell stage embryos, but not in oocytes, from Slc6a19 -/- mice compared to those from WT mice. Other B 0 AT1 substrates, such as alanine and leucine, reduced uptake of Pro in WT but not in B 0 AT1 knockout embryos. Addition of Pro to culture medium improved embryo development. In WT embryos, Pro increased development to the cavitation stage (on day 4); whereas in B 0 AT1 knockout embryos Pro improved development to the 5-8-cell (day 3) and blastocyst stages (day 6) but not at cavitation (day 4), suggesting B 0 AT1 is the main contributor to Pro uptake on day 4 of development. Our results highlight transporter redundancy in the preimplantation embryo.