Fertilization and Early Embryogenesis
(M283) THE ORGANIC OSMOLYTES BETAINE AND PROLINE ARE TRANSPORTED BY A NOVEL SHARED TRANSPORT PATHWAY IN EARLY MOUSE EMBRYOS.
Anas, Idris1, Hammer, Mary-Anne 1, Baltz, Jay1, 1 University of Ottawa and the Ottawa Health Research Institute, Ottawa, ON, Canada
ABSTRACT- Betaine and proline protect mouse embryos against increased osmolarity, functioning as organic osmolytes. However, their transport in early embryos is not mediated by any of the four known mammalian organic osmolyte transporters, and, although we recently identified the glycine transporter GLYT1 as a fifth organic osmolyte transporter apparently unique to early embryos, neither betaine nor proline are substrates. Here, we examined the unidentified betaine and proline transporter in 1-cell mouse embryos. Betaine and proline transport were∼90% saturable. However, whereas ∼90% of betaine transport was inhibited by unlabeled proline, maximal inhibition of proline uptake by betaine was only ∼70%, suggesting that proline is also transported by a second betaine-insensitive system. This component was Na+-dependent and inhibited by alanine, leucine, cysteine and methionine, suggesting an ASC amino acid transporter-like mechanism. We then investigated the shared betaine and proline transport system (with betaine-insensitive proline transport eliminated by excess alanine), measuring Kms of ∼200 and ∼300 &uscript;M for betaine and proline transport, respectively, with a Vmax of ∼3-4 fmol/embryo/min. Transport was Na+- and Cl--dependent. Betaine and proline inhibited each other with Kis of ∼125 &uscript;M for proline and ∼200 &uscript;M for betaine, close to their own Kms. Transport of both was completely inhibited by several proline derivatives, and partially by dimethylglycine, sarcosine, phenylalanine, norleucine and cysteine. More detailed measurements with dimethylglycine revealed a similar Ki against both betaine and proline of ∼2 mM. These findings indicate that 1-cell mouse embryos possess a single shared betaine and proline transporter that is likely a volume-regulatory organic osmolyte transporter, while some proline is also transported by a second ASC-like transport system. Supported by the Canadian Institutes of Health Research (MOP62730), and a Lalor Foundation fellowship (IA).
KEY WORDS: organic osmolytes, early embryo development, Betaine, Proline