Stage-Specific L-Proline Uptake by Amino Acid Transporter Slc6a19/B0AT1 Is Required for Optimal Preimplantation Embryo Development in Mice

Abstract

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.

Proline and Proline Analogues Improve Development of Mouse Preimplantation Embryos by Protecting Them against Oxidative Stress

“…It is also not known at this stage if any of the Pro-mediated mechanisms that improve development at the late 2-cell to 8-cell stage are required to maintain that developmental advantage out to the hatching blastocyst stage. What does appear to be clear, however, is that Pro acts at various points in development from the oocyte to the production of neurectoderm, consistent with its growth factor-like properties [4,38,39,41,52].…”

“…Pro is a conditional non-essential AA whose concentration in follicular and oviductal fluid across a number of mammalian species ranges from 0.05-0.6 mM [42][43][44][45]. AA transporters for Pro are present throughout development of the pre-implantation embryo [40,[46][47][48][49][50][51][52]. When 400 µM Pro is added to culture medium, it is taken up into the embryo to millimolar concentrations [46] and improves in vitro embryo development in a growth factor-like manner, from the late 2-to 8-cell stage out to the blastocyst stage, including hatching [38].…”

Molecular and Clinical Advances in Understanding Early Embryo Development

“…Remarkably, our knowledge of the importance of amino acid transport, signaling, and metabolism to preimplantation development continues to develop. For example, in a paper of this Special Issue, Treleaven et al report how proline transport into preimplantation mouse embryos fosters their development but in a stage-specific manner [ 5 ]. In other words, proline improves the preimplantation development of mouse zygotes, and the B 0 amino acid transporter appears to be mainly responsible for this uptake on day 4 but not on other days of development [ 5 ].…”

“…For example, in a paper of this Special Issue, Treleaven et al report how proline transport into preimplantation mouse embryos fosters their development but in a stage-specific manner [ 5 ]. In other words, proline improves the preimplantation development of mouse zygotes, and the B 0 amino acid transporter appears to be mainly responsible for this uptake on day 4 but not on other days of development [ 5 ]. Moreover, proline fosters development in a growth-factor-like manner, in part because proline can substitute for the apparent paracrine signaling among these embryos grown at a high density [ 1 , 6 ].…”

“…Of the amino acids, proline seems to be most beneficial for preimplantation development in vitro. Therefore, the most effective way to culture healthy mouse and possibly human embryos is to supply only proline in these media [ 5 , 6 ]. Although complex, conditions in vivo can also be highly suitable for fostering the development of the healthiest preimplantation embryos under physiologically normal conditions [ 1 , 7 ].…”