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

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.

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.

Australian Public Attitudes on Gene Editing of the Human Embryo.

Technology is now available which facilitates gene editing and has recently been applied internationally to embryos in the laboratory. A 2002 law in Australia prohibits making heritable changes in embryos, regardless of whether the treated embryo is discarded thereafter. We sought to begin to understand public opinion in Australia about this matter, using a questionnaire given to the audience attending a Q and A panel of experts. We found majority support for allowing heritable changes for health purposes. If this is confirmed in a larger survey of the population, we suggest the existing law should be reviewed.

Predicting Public Attitudes Toward Gene Editing of Germlines: The Impact of Moral and Hereditary Concern in Human and Animal Applications.

Background and Objective: New and more efficient methods of gene editing have intensified the ethical and legal issues associated with editing germlines. Yet no research has separated the impact of hereditary concern on public attitudes from moral concern. This research compares the impact these two concerns have on public attitudes across five applications including, the prevention of human disease, human and animal research, animals for the use of human food and the enhancement of human appearance. Methods: A sample of 1004 Australians responded to either a telephone (n = 501; randomly selected) or online survey (n = 503; sourced by Qualtrics). Both samples were representative in terms of States and Territories as well as gender (51% female), though the online sample was younger (M = 40.64, SD = 16.98; Range = 18-87) than the telephone sample (M = 54.79, SD = 18.13; Range = 18-96). A 5 (application) by 3 (type of cell) within groups design was utilized, where all respondents reported their level of approval with scientists editing genes across the 15 different contexts. Multilevel modeling was used to examine the impact of moral (embryo vs. germ) and hereditary (germ vs. somatic) concern on attitudes across all applications. Results: Australians were comfortable with editing human and animal embryos, but only for research purposes and to enhance human health. The effect of moral concern was stronger than hereditary concern, existing in all applications except for the use of animals for human purposes. Hereditary concern was only found to influence attitudes in two applications: improving human health and human research. Moral concern was found to be accentuated amongst, women, more religious individuals and those identifying as Australian, while hereditary concern was strongest amongst non-Australians, those with stronger trust in scientists, and more religious respondents. Conclusion: Moral and hereditary concerns are distinct, and require different approaches to public education, engagement and possibly regulation. Further research needs to explore hereditary concern in relation to non-human applications, and the reasons underlying cultural and gender differences.