Engineering Stress-Tolerant Mammalian Embryos via a Functional Trehalose Transporter.

Trehalose is a disaccharide known for protecting cells against desiccation and freezing. However, mammalian cells lack endogenous trehalose transporters, limiting its intracellular utility. In this study, we examined whether transient expression of pvTret1, a trehalose transporter from the desiccation-tolerant midge Polypedilum vanderplanki, could enable trehalose uptake in mouse embryos without impairing development. We synthesized mRNA encoding pvTret1-eGFP and microinjected it into mouse zygotes. Fluorescence microscopy confirmed plasma membrane localization, and AlphaFold modeling indicated that eGFP fusion did not alter protein structure. Embryos expressing pvTret1 developed to the blastocyst stage and produced live offspring at rates comparable to sham-injected controls, suggesting that transient expression does not compromise developmental potential. LC-MS analysis revealed that pvTret1-expressing embryos took up trehalose in a concentration-dependent manner following 20-minute exposure to trehalose-containing medium, and effectively cleared intracellular trehalose within 20 minutes of removal, indicating functional, bidirectional transport. In contrast, control embryos exhibited shrinkage under hyperosmotic conditions. Embryos with intracellular trehalose also showed improved post-warming survival after vitrification in DMSO-free, trehalose-based cryoprotectant solutions. Although optimization is ongoing, these findings highlight the potential utility of pvTret1-mediated trehalose uptake in developing low-toxicity embryo preservation methods. This is the first demonstration that pvTret1 can function in mammalian embryos, enabling transient intracellular trehalose accumulation without transgenesis. Future work will focus on refining trehalose loading protocols and advancing vitrification or dry-preservation strategies for reproductive and biomedical applications. .