Morris, M. B. | bioRxiv Science

bioRxiv2026

Culture of preimplantation embryos in media containing L-proline increases intracellular GSH concentration throughout development

Careful balance of the redox status of the embryo and reduction of oxidative stress is crucial in early development. Here we show that the culture of preimplantation mouse embryos in the conditionally non-essential amino acid L-proline (Pro) increases the intracellular concentration of the potent antioxidant glutathione as shown by staining of 2-cell, 4-cell and 8-cell embryos with tetrafluoroterephthalonitrile (4F-2CN). Further, liquid-chromatography/mass spectrometry showed increased GSH levels in all Pro-treated preimplantation stages of development compared to controls. The GSH:GSSG ratio also showed a Pro-dependent increase. Overall, our results indicate that the beneficial effect of Pro in preimplantation embryo culture is due to the reduction in oxidative stress mediated through an increase in cellular GSH concentration.

cell biology↗

bioRxiv2023

Signalling pathway crosstalk stimulated by L-proline drives differentiation of mouse embryonic stem cells to primitive-ectoderm-like cells

The amino acid L-proline exhibits novel growth factor-like properties during development - from improving blastocyst development to driving neurogenesis in vitro. Addition of 400 M L-proline to self-renewal medium drives mouse embryonic stem cells (ESCs) to a transcriptionally distinct pluripotent cell population - early primitive ectoderm-like (EPL) cells - which lies between the naive and primed states. EPL cells retain expression of pluripotency genes, upregulate primitive ectoderm markers, undergo a morphological change and have increased cell number. These changes are facilitated by a complex signalling network hinging on the Mapk, Fgfr, Pi3k and mTor pathways. We use a factorial experimental design coupled with linear modelling and Bayesian regularised neural networks to understand which signalling pathways are involved in the transition between ESCs and EPL cells, and how they underpin changes in morphology, cell number, apoptosis, proliferation and gene expression. This approach allows for consideration of where pathways work antagonistically or synergistically. Modelling showed that most properties were affected by more than one inhibitor, and each inhibitor blocked specific aspects of differentiation. These mechanisms underpin both progression of stem cells across the in vitro pluripotency continuum and serve as a model for pre-, peri- and post-implantation embryogenesis. Summary StatementL-proline acts as growth factor to modulate phosphorylation of the Mapk, Pi3k, Fgf and mTor signalling pathways to drive embryonic stem cells to primitive ectoderm-like cells.

developmental biology↗