Abstract / Summary
PurposeThis study provides preliminary transcriptomic insights into the divergent responses of pseudo-mosaic and uniform aphidicolin-treated embryos, highlighting the need for further mechanistic studies and singleembryo-level analysis to validate these observations. MethodsFollowing a preliminary experiment evaluating prolonged aphidicolin exposure, an independent cohort of 90 four-cell cryopreserved mouse embryos was used for the main experiment and randomly allocated to three groups (n = 30 each).Control embryos received no intervention.In the pseudo-mosaic group, two blastomeres were removed from each embryo, exposed to 1.4 µM aphidicolin for four hours, and reinjected into their corresponding embryo.In the uniform aphidicolin-treated group, intact embryos were exposed to 1.4 µM aphidicolin for four hours.Blastocyst formation was assessed, and RNA sequencing was performed to compare differential gene expression and pathway enrichment among the three groups. ResultsIn the initial study, prolonged exposure (16 hours) to 1.4 µM aphidicolin resulted in complete cleavage-stage arrest in the uniform aphidicolin-treated group.Following four hours of aphidicolin exposure in the main study, blastocyst formation rates were not significantly different among groups: control, 26/30 (87%); pseudo-mosaic, 22/30 (73%); and uniform aphidicolin-treated, 27/30 (90%) (p > 0.05).When the control group was compared with the uniform aphidicolin-treated group, 33 genes were differentially expressed at an adjusted p-value of <0.05.When the control group was compared with the pseudo-mosaic group, 791 genes were differentially expressed at an adjusted p-value of <0.05.Gene set enrichment analysis comparing uniform aphidicolin-treated and pseudo-mosaic embryos identified divergent pathway enrichment profiles, ranging from cell-cycle and transcriptional regulation in the former group to cell-fate reprogramming and metabolic adaptation in the latter. ConclusionsThis study reveals distinct transcriptomic responses to aphidicolin-induced replication stress in pseudomosaic versus uniform aphidicolin-treated embryos.The findings suggest that embryos containing discordant cell populations may engage compensatory transcriptional programs that differ from those activated following uniform cellular stress.While the observed pathway profiles are consistent with DNAdamage, stress-response, metabolic, and apoptotic processes, the absence of genomic validation limits interpretation.Future studies incorporating single-embryo analysis and genomic characterization are needed to confirm these preliminary observations.