L26/P-154 Spermidine improves developmental competence in embryos from aged mice by enhancing autophagy during zygotic genome activation

Abstract Study question Can spermidine supplementation improve developmental potential in embryos from aged mice by promoting autophagy during zygotic genome activation (ZGA)? Summary answer Spermidine supplementation enhances autophagy and restores maternal transcript clearance in embryos from aged mice, resulting in improved development and outgrowth capacity. What is known already Autophagy is essential for early embryonic development and cytoplasmic remodeling after fertilization. We have previously demonstrated that reduced autophagy at the 2-cell stage in embryos from aged mice is associated with impaired maternal mRNA clearance and disrupted ZGA. Spermidine, a natural polyamine, is a well-established autophagy inducer with anti-aging properties. We therefore investigated whether spermidine supplementation could enhance autophagy during ZGA, restore maternal transcript clearance, and improve developmental competence in embryos from aged mice. Study design, size, duration This study examined the effects of spermidine supplementation on embryos from aged mice. An initial dose-screening identified 25 μM spermidine as the optimal concentration based on fertilization and blastocyst formation rates. MII oocytes from aged B6D2F1 females were fertilized and cultured with or without spermidine. Outcomes were assessed at molecular and developmental levels, focusing on events at the 2-cell stage and subsequent developmental competence. Participants/materials, setting, methods MII oocytes were collected from aged (65–80 weeks) B6D2F1 female mice and fertilized in medium supplemented with or without spermidine. Embryos were cultured under identical conditions. Autophagy at the 2-cell stage was assessed by LC3 immunofluorescence and Cyto-ID staining. Maternal transcript clearance was evaluated by qPCR for Bmp15 and Gdf9. Developmental competence was assessed by blastocyst formation on Day 4 and quantified blastocyst outgrowth area using an in vitro attachment assay. Main results and the role of chance Initial dose screening was performed in embryos from aged mice using 0, 12.5, 25, and 50 μM spermidine. The 12.5 μM dose showed minimal effects, whereas 50 μM impaired embryonic development. In contrast, 25 μM showed the highest blastocyst formation and the lowest degeneration among all tested concentrations. This concentration was therefore selected for use in all subsequent experiments. Spermidine supplementation significantly enhanced autophagy activity in embryos from aged mice, as evidenced by increased LC3-positive puncta and elevated Cyto-ID signal at the 2-cell stage. Moreover, spermidine treatment restored appropriate maternal transcript clearance during the ZGA, as demonstrated by normalized expression dynamics of Bmp15 and Gdf9. Embryos exposed to spermidine from the fertilization stage showed significantly higher 2-cell cleavage and blastocyst formation rates compared with untreated controls, whereas the 4-cell development rate was increased but did not reach statistical significance. In addition, blastocysts cultured with spermidine demonstrated significantly increased outgrowth area in vitro, indicating enhanced implantation potential. Collectively, these results demonstrate that spermidine supplementation promotes ZGA-associated autophagy, facilitates maternal transcript clearance, and improves developmental competence in embryos from aged mice. Limitations, reasons for caution This study was conducted in a murine model. Although these findings support a role for spermidine-induced autophagy during ZGA in embryos from aged mice, validation in human embryos is required before clinical application can be considered. Wider implications of the findings These findings suggest that spermidine-based modulation of autophagy during the ZGA may represent a potential approach for improving embryo quality associated with reproductive aging. This strategy may also inform optimization of embryo culture conditions in assisted reproductive technologies. Trial registration number No